Method for producing high-purity and highly stable proteins

Abstract

The present invention provides a method for obtaining highly pure and stable recombinant human albumin by adding a medium- to long-chain fatty acid ligand and removing charge-heterogeneous proteins through anion and / or cation chromatography. Uniquely, the method includes the steps of: preparing a mixture of a fatty acid mixture and a poloxamer, in which the molar ratio of oleic acid, myristic acid, sodium palmitate, sodium stearate, and recombinant human albumin is 0.3:0.3:0.3:0.5:1; and sequentially performing ion exchange chromatography to effectively remove cleaved albumin, mismatched albumin, or modified albumin from the recombinant albumin, thereby obtaining charge-heterogeneity-removed albumin and modified albumin, thereby obtaining a highly pure and stable human albumin sample.

Description

Technical Field

[0001] The present invention is a method for obtaining highly pure recombinant human albumin by adding a medium-chain and long-chain fatty acid ligand and removing charge inhomogeneity by anion or / and cation chromatography.

Background Art

[0002] The main pharmacological actions of human albumin are to regulate the dynamic balance of water between tissues and blood vessels, maintain a normal and constant plasma volume, have a high affinity for certain ions and compounds, reversibly bind to these substances to exert a transfer function, and further store a large amount of amino acids for the living body.

[0003] Due to the above actions of human albumin, it can be applied to various clinical subjects to exert various therapeutic effects. Clinically, human albumin mainly adjusts the colloid osmotic pressure of plasma, expands blood volume, treats traumatic and hemorrhagic shock, severe burns and hypoproteinemia, and is widely applied in general diseases such as stroke, liver cirrhosis, hepatic ascites and kidney diseases. In addition to being directly applied in the field of clinical treatment, albumin is also very widely applied in multiple aspects such as the medium used in vaccine production, pharmaceutical adjuvants, diagnostic reagents, novel long-acting tumor preparation products, cosmetics, experimental biological reagents, etc.

[0004] The structure of human albumin is a single-chain non-glycosylated protein with a heart-shaped structure, having 585 amino acids, 17 pairs of disulfide bonds, and 1 free thiol group, and a molecular weight of 66,438 daltons. The half-life of human albumin in the human body is 19 to 21 days. The heart-shaped structure of human albumin is composed of three main domains and six subdomains wrapped by 17 disulfide bonds, which are loosely bound by van der Waals forces. As can be seen from its crystal structure, the disulfide bridges give rigidity to the helical globular structure, but provide sufficient flexibility for the protein to undergo structural changes in response to changes in the surrounding medium.

[0005] Human albumin is generally produced by extracting, separating, and purifying human serum, and is collectively referred to as human serum albumin. Human albumin derived from human blood poses significant risks in clinical use due to limitations in the quantity of plasma sources, viral contamination by plasma donors, and differences in individual antibodies and proteins. Therefore, the instructions for use of human serum albumin in many countries all include a statement regarding viral safety, for example, that "standard measures taken to prevent infection from the use of human blood or plasma products include donor selection, screening of single donated blood or plasma pools for special infection marks, and the use of effective manufacturing steps for viral inactivation / removal. Nevertheless, when using pharmaceutical products produced from blood or plasma, the possibility of infection by infectious pathogens cannot be ruled out. This includes unknown or newly emerging viruses and other pathogens." Therefore, the use of genetic engineering methods is the optimal route to effectively obtain albumin free from viral contamination.

[0006] Currently, the most common method for mass-producing recombinant human albumin through microbial expression is primarily yeast expression systems, with budding yeast and Pichia yeast being the most mature. However, since human albumin is a high-volume injectable drug, each injection can reach 5-30 g. Therefore, the overall host protein residue in each injection and contaminants during the manufacturing process must be kept below 1 ng / ml (200 mg / ml-rHA). Regardless of the method used to produce recombinant human albumin, modified recombinant albumin, such as glycosylation, oxidation, macromerization, mismatching, and aggregation, can potentially cause immunotoxicity in the human body and may have some impact on protein stability. Therefore, efficient and specific purification methods are crucial elements in the process of obtaining highly pure, homogenized, and uniform recombinant human albumin.

[0007] Patent WO1966 / 037515 (CN1198844C) discloses a method for adding a medium-short chain fatty acid such as sodium octanoate before ion-exchange chromatography, which can remove to some extent charge heterogeneous albumin after glycosylation modification. However, sodium octanoate has weak ligand activity and cannot effectively remove charge heterogeneous albumin that is mismatched, oxidized, and cleaved.

[0008] Patent CN2019108743433 discloses a method for adding medium- and long-chain fatty acids or salts and trace amounts of polymer additives to recombinant albumin preparations to improve the stability of the albumin preparations and prevent aggregation and aggregation. However, this method cannot selectively remove hydrophobic heterogeneity and charge heterogeneity deficiencies, isomers, or post-modified albumin, and cannot remove unstable small amounts of heterogeneous albumin from the outset.

[0009] The present invention provides a reasonable ligand ratio for albumin by adding a certain amount of medium- and long-chain fatty acids and fatty acid salts before purified ion-exchange chromatography, thereby assisting in the effective spatial folding of albumin and exhibiting charge heterogeneous albumin and charge heterogeneous spaces. Under these conditions, ion-exchange chromatography can be used to effectively remove albumin with inaccurate or post-modified structures, such as fragments and mismatches, generated during the fermentation and purification of recombinant albumin.

[0010] In this specification, the term “recombinant human albumin” may also be referred to as “recombinant albumin” and / or “recombinant human serum albumin” and / or “recombinant human blood albumin” and / or “rHA” and / or “rHSA”. The term “human serum albumin” refers to human albumin extracted from human serum and may also be referred to as “human blood albumin” and / or “HSA” and / or “HA” and / or “pdHSA”. In this specification, the term “medium- and long-chain fatty acids” means natural fatty acids and their salts having a carbon chain of more than 10. [Overview of the project] [Problems that the invention aims to solve]

[0011] Purpose of the invention To provide a method for producing high-purity and highly stable proteins with superior effectiveness, we will refer to several substantial technical effects of specific implementations for specific purposes. [Means for solving the problem]

[0012] To achieve the above objective, the technical means used in the present invention are as follows.

[0013] A method for producing high-purity and highly stable proteins is: The recombinant human albumin sample collected by the previous purification process was placed in a Millipore 10kDa membrane pack and the buffer system was changed to one with 80 mM PB of buffer at pH 7.5. The mixed solution was added to the sample collection solution, and the mixed solution was made into a mixture of fatty acid mixture and poloxamer, with a molar ratio of oleic acid, myristic acid, sodium palmitate, sodium stearate, and recombinant human albumin of 0.3:0.3:0.3:0.5:1. Step A involves injecting the sample, washing the anion exchange chromatography column with purified water to neutralize it, packing the chromatography column to a height of 380 mm, eluting the chromatography column with the sample adsorbed using a 50 mM PB pH 6.5 eluent system, and collecting the recombinant human albumin component. The method is characterized by comprising step B, in which the chromatography column is equilibrated with a 40 mM HAc-NaOH equilibrium liquid system in cation exchange chromatography, the collected recombinant human albumin components are desalted with a Millipore 10 kDa flat membrane and replaced with the cation exchange chromatography equilibrium liquid system, and then sample injection and chromatographic separation are performed to collect the recombinant human albumin components.

[0014] A method for producing high-purity and highly stable proteins is: Step C involves replacing the recombinant human albumin sample collected by the previous purification process with an equilibrium liquid system of 40 mM HAc-NaOH buffer in a Millipore 10kDa membrane pack, equilibrating the cation exchange chromatography column with the equilibrium solution, adding the mixture to the sample, making the mixture a mixture of fatty acid mixture and poloxamer, with a molar ratio of oleic acid, myristic acid, sodium palmitate, sodium stearate, and recombinant human albumin of 0.3:0.3:0.3:0.5:1, followed by sample injection and chromatographic separation to collect the recombinant human albumin components. Step D is characterized by comprising: replacing the buffer system of Step A with the recombinant human albumin components collected in Step C using a Millipore 10kDa membrane pack; equilibrating the anion exchange chromatography column with the equilibrium solution of Step A; and collecting the recombinant human albumin components by performing the sample injection and purification in the same manner as in Step A.

[0015] As a further technical means of the present invention, after step B and / or step D, when the chromatography is finally completed, the step of removing and replacing the polymer aggregate fraction and small molecule substances using a 100 kDa and / or 30 kDa and / or 10 kDa membrane pack is further included to concentrate the recombinant human albumin stock solution to a concentration greater than 20%.

[0016] As a further technical means of the present invention, the cation exchange medium includes, but is not limited to, SP strong cation or CM weak cation exchange medium, and the cation exchange medium may include, but is not limited to, the Uni-SP / CM series, UniGel-SP / CM series, NanoGel-SP series, Nano-SP series, Monomix-HC SP series, Monomix-MC SP series, GE's Sepharose series, or BestChrom Biotechnology Co., Ltd.'s Beststarose series, and the anion exchange medium includes, but is not limited to, Q strong anion or DEAE weak anion exchange medium, and the anion exchange medium may include, but is not limited to, the Uni-DEAE / Q series, UniGel-DEAE / Q series, NanoGel-Q series, Nano-Q series, Monomix-HC DEAE / Q series, Monomix-MC DEAE / Q series, GE's Sepharose series, or BestChrom Biotechnology Co., Ltd.'s Beststarose series, and is not limited to these.

[0017] As a further technical means of the present invention, the weight ratio of poloxamer to recombinant albumin is set to 10 μg to 500 μg of poloxamer / 1 g of recombinant albumin, and poloxamer is used to promote fatty acid dissolution.

[0018] As a further technical means of the present invention, the anion exchange chromatography is a purification by forward elution, in which the pH of both the equilibrium solution and the eluent is 6.0 to 9.5, and in the method of the present invention, the electrical conductivity under the conditions of anion exchange chromatography is 20 ms / cm or less, or the anion exchange chromatography is a purification by reverse elution, in which the pH of the equilibrium solution is 4.0 to 6.0, and the equilibrium electrical conductivity under the conditions of anion exchange chromatography is 10 ms / cm or less.

[0019] As a further technical means of the present invention, anion exchange chromatography is purification by a forward elution method. Preferably, the pH of both the equilibration solution and the elution solution is 6.5 to 9.0. Preferably, the electrical conductivity under the conditions of anion exchange chromatography is 15 ms / cm or less. Alternatively, anion exchange chromatography is purification by a reverse elution method. Preferably, the pH of the equilibration solution is 4.0 to 5.5. Preferably, the equilibrium electrical conductivity under the conditions of anion exchange chromatography is 6 ms / cm or less.

[0020] As a further technical means of the present invention, the pH of the equilibration solution under the conditions of cation exchange chromatography is 4.0 to 6.5, and the electrical conductivity under the conditions of cation exchange chromatography is 10 ms / cm or less.

[0021] As a further technical means of the present invention, preferably, the pH of the equilibration solution under the conditions of cation exchange chromatography is 4.5 to 6.0, and preferably, the electrical conductivity under the conditions of cation exchange chromatography is 5 ms / cm or less.

[0022] As a further technical means of the present invention, the recombinant human albumin sample collected after the previous purification is human albumin expressed by genetically recombinant microorganisms produced on a large scale.

Effects of the Invention

[0023] Compared with the prior art, the present invention using the above technical means has the following beneficial effects. As the unique first point of the present invention, the molar ratio of oleic acid, myristic acid, sodium palmitate, sodium stearate and recombinant human albumin is 0.3:0.3:0.3:0.5:1, that is, the molar ratio of long-chain fatty acids and fatty acid salts: human recombinant albumin is 0.1 to 3:1, and sequentially using ion exchange chromatography can effectively remove cleaved albumin, mismatched or modified albumin in recombinant albumin, and these components may clinically cause side effects to the human body or affect the half-life and drug efficacy.

[0024] As the unique second point of the present invention, by adding medium-chain and long-chain fatty acids and fatty acid salts before purified ion exchange chromatography, the ratio of effective fatty acid ligands can be selectively retained by ion exchange, and components with too high or too low ligand ratios after hydrophobic inhomogeneity occurs due to modification or folding error of the albumin structure can be removed by ion exchange, and a human albumin sample with high purity and high stability can be obtained.

Brief Description of the Drawings

[0025] To further illustrate the present invention, the following will be further described with reference to the drawings.

[0026] [Figure 1] It is a comparison diagram of the charge inhomogeneity detection spectrum of the sample before performing Example 1 and the charge inhomogeneity detection spectrum of the sample after sequentially performing the purification of Example 1 and Example 2. The vertical axis of FIG. 1 is the absorption value (mAU), and the horizontal axis is the time axis (min). [Figure 2] It is the detection of the particle size distribution status of the measurement sample at 0 hour, 1 month, and 3 months by DLS (dynamic light scattering meter) in the accelerated stability experiment. The vertical axis of FIG. 2 is the distribution percentage, and the horizontal axis is the molecular diameter (nm). [Figure 3]This figure shows the particle size distribution of a sample measured using DLS (Dynamic Light Scattering) in a long-term stability experiment at 0:00, 12 months, 24 months, and 36 months. In Figure 3, the vertical axis represents the distribution percentage, and the horizontal axis represents the molecular diameter (nm). [Modes for carrying out the invention]

[0027] The present invention will be further described below with reference to the drawings and specific embodiments. Please understand that the following specific embodiments are merely for illustrative purposes and do not limit the scope of the present invention.

[0028] The fermentation scales of the present invention were carried out in 10L, 20L, 3000L, and 100000L facilities, and the purification scales were carried out in column diameters of 10cm, 45cm, and 120cm, respectively, and the linear expansion was good. This example includes, but is not limited to, the above scales. The following examples are for further understanding the features and advantages of the present invention with reference to the drawings and do not limit other content that may be interpreted from the present invention in any way.

[0029] (Example 1) Anion exchange chromatography The recombinant human albumin sample collected by the previous purification process was replaced with an equilibrium liquid system of 80 mM PB buffer at pH 7.5 using a Millipore 10 kDa membrane pack. A mixture of oleic acid, myristic acid, sodium palmitate, sodium stearate, and poloxamer was added to the sample collection solution to achieve a final fatty acid concentration of 0.3:0.3:0.3:0.5:1 (oleic acid: myristic acid: sodium palmitate: sodium stearate: recombinant human albumin). After injecting the sample, the UniGel-DEAE chromatography column (packed to a height of 380 mm) was washed to neutral with purified water. The chromatography column with the adsorbed sample was eluted using a 50 mM PB pH 6.5 eluent system to collect the recombinant human albumin component.

[0030] (Example 2) Cation exchange chromatography For cation exchange chromatography, SP Bestarose FF filler from BestChrom (Shanghai) Biotechnology Co., Ltd. (this filler is equivalent to GE SP Sepharose Fast Flow filler) was used. The chromatography column was equilibrated with a 40 mM HAc-NaOH equilibrium liquid system, and the recombinant human albumin solution collected from Example 1 was desalted using a millipore 10 kDa flat membrane and replaced with the equilibrium liquid system. Then, the sample was injected and chromatographic separation was performed to collect the components of recombinant human albumin.

[0031] (Example 3) Exchange between cation exchange chromatography and anion exchange chromatography In this example, first, Example 2 was carried out, and the recombinant human albumin sample collected after initial purification was replaced in the buffer system of Example 2 using a Millipore 10kDa membrane pack. A mixture of oleic acid, myristic acid, sodium palmitate, sodium stearate, and poloxamer was added to replenish the system, and the final concentrations of the fatty acids were adjusted to a molar ratio of 0.3:0.3:0.3:0.5:1 (oleic acid: myristic acid: sodium palmitate: sodium stearate: recombinant human albumin). The recombinant human albumin component was collected using the same method as in Example 2.

[0032] The collected recombinant human albumin components were replaced in the equilibrium liquid system of Example 1 using a Millipore 30kDa membrane pack, and column equilibrium was performed. Purification, sample injection, and collection of recombinant human albumin components were then carried out in the same manner as in Example 1.

[0033] When the above chromatography is finally completed, the polymer aggregates and small molecule substances can be removed and replaced using 100 kDa and / or 30 kDa and / or 10 kDa membrane packs to concentrate the recombinant human albumin stock solution to a concentration greater than 20%. Charge heterogeneity was detected in the sample before performing Example 1 and in the sample after performing Examples 1 and 2.

[0034] The method for detecting charge heterogeneity was based on ion chromatography, as described in General Rules 0513 of the Fourth Section of the Chinese Pharmacopoeia, 2020. The detection results for recombinant human albumin are as follows (Figure 1).

[0035] (Example 4) Acceleration stability experiment In this example, the protein solution purified in Example 2 was used to complete the production of the stock solution. Recombinant human albumin injection was then prepared, and the injection sample was subjected to accelerated testing under conditions of 25°C ± 2°C and 60% ± 5% humidity. The study was generally conducted for up to 6 months, and the measurement results (Tables 1 and 2) and diagrams (Figure 1) are shown.

[0036] (Example 5) Long-term stability experiment In this example, the protein solution purified in Example 2 was used to complete the production of the stock solution. After that, recombinant human albumin injection was prepared, and the injection sample was subjected to long-term stability measurements under conditions of 5°C ± 3°C. For the analysis, detection was generally performed every 3 months in the first year, for example, at 0, 3, 6, 9, and 12 months. In the following year, detection was performed every 6 months, for example, at 18 and 24 months. Thereafter, detection was performed annually, for example, at 36 months. The measurement results (Tables 3 and 4) and the diagram (Figure 3) are shown.

[0037] Table 1 Detection Results [Table 1]

[0038] Table 2: T of the sample measured by DSC at 0 and 3 months. onset , T m1 , T m2 A change was detected. [Table 2]

[0039] The particle size distribution of the measurement sample at 0:00, 1 month, and 3 months was detected using DLS in Figure 2, and no significant changes were observed in the particle size distribution of the sample during the accelerated stability experiment.

[0040] Table 3 Detection Results [Table 3]

[0041] Table 4. T of the sample measured by DSC at 0 and 36 months. onset , T m1 , T m2 A change was detected. [Table 4]

[0042] Figure 3 shows that DLS detected the particle size distribution of the measurement sample at 0:00, 12 months, 24 months, and 36 months. Long-term stability experiments showed no significant changes in the particle size distribution of the sample.

[0043] In short, the present invention provides a method for removing proteins with incorrect structures, such as fragments and mismatches generated during the fermentation and purification of recombinant albumin, by adding a certain amount of medium- and long-chain fatty acids and fatty acid salts under the conditions of a recombinant albumin purification intermediate, and using ion exchange chromatography. The method of the present invention involves purifying the purified recombinant human albumin solution by means of cation exchange chromatography, anion exchange chromatography, etc.

[0044] 1) Anion exchange chromatography 2) Cation exchange chromatography

[0045] The cation exchange medium of the present invention is the SP series, or the cation exchange medium of the CM series may include, but is not limited to, the Uni-SP / CM series, UniGel-SP / CM series, NanoGel-SP series, Nano-SP series, Monomix-HC SP series, Monomix-MC SP series, GE's Sepharose series, or BestChrom Biotechnology Co., Ltd.'s Bestarose series.

[0046] The medium for the anion exchange chromatography of the present invention is the DEAE series or the Q series of strong anions. The anion exchange medium may include, but is not limited to, the Uni-DEAE / Q series, UniGel-DEAE / Q series, NanoGel-Q series, Nano-Q series, Monomix-HC DEAE / Q series, Monomix-MC DEAE / Q series, GE's Sepharose series, or BestChrom Biotechnology Co., Ltd.'s Bestarose series.

[0047] Long-chain fatty acids may be added before anion exchange chromatography or supplemented before cation exchange chromatography.

[0048] The replacement and concentration of buffers during the chromatography steps of the present invention can be carried out using devices and equipment such as hollow fiber membranes and flat membrane packs with separation pore sizes between 1 kDa and 30 kDa molecular weight, and such implementation includes, but is not limited to, the order of use and cross-use.

[0049] The buffer substitution during the chromatography steps of the present invention may be performed using Sephadex G25 or Superdex G75.

[0050] Recombinant albumin preparations with high charge matching and hydrophobic matching obtained after carrying out the present invention can effectively meet the stability requirement of being left at 57°C for 50 hours according to the formulations of the Chinese Pharmacopoeia (2020 edition) or the United States Pharmacopeia (USP39), and can also be stored stably for 30 days or more under conditions of 25°C or 30°C.

[0051] The poloxamer described in the present invention is one or more selected from poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407.

[0052] The high-purity recombinant human albumin obtained by the method of the present invention can be applied to clinical indications that are the same as those of serum albumin derived from human blood.

[0053] The high-purity recombinant human albumin obtained by the method of the present invention can be used in diagnostic reagents, culture media, and medicinal adjuvants.

[0054] The separation medium used in the present invention includes, but is not limited to, an ion exchange medium of hydrophilic modified grafted microspheres measuring 10 μm to 150 μm, made of polyacrylate or polystyrene-divinylbenzene polymer. It may also be Sepharose 6 FF, or any one of the two polymer substrates or agarose matrix having the length of the grafted side chains.

[0055] In this invention, the column bed is filled to a height of 100 mm to 800 mm. Preferably, it is 250 mm to 600 mm.

[0056] The chromatography conditions described in this invention can be adjusted and modified according to a general manual.

[0057] During the chromatography described in the present invention, steps such as dialysis, ultrafiltration, and low-temperature inactivation may be performed using several other conventional methods without affecting the effectiveness of the present invention.

[0058] After the chromatography of the present invention is finally completed, the polymer aggregates can be fractionated and small molecule substances removed using a 100 kDa and / or 30 kDa and / or 10 kDa membrane pack, and then replaced to concentrate the recombinant human albumin stock solution to a concentration higher than the formulation concentration.

[0059] The expression host cells described in the present invention are yeasts including budding yeasts of the genus Saccharomyces, yeasts of the genus Kluiveromyces, yeasts of the genus Hansenula, and yeasts of the genus Pichia.

[0060] The basic principles, main features, and advantages of the present invention have been described above. Those skilled in the art will understand that the present invention is not limited to the above embodiments, and that the above embodiments and specification are merely for illustrating the principles of the present invention. The present invention may also have various variations and improvements without departing from the spirit and scope of the invention, and all such variations and improvements will be within the scope of the claims.

Claims

1. The recombinant human albumin sample collected by the previous purification process was replaced with an equilibrium liquid system of 80 mM phosphate buffer at pH 7.5 using a 10 kDa membrane module. A mixture was added to the sample collection solution, and the mixture was prepared as a mixture of fatty acid mixture and poloxamer. The molar ratio of oleic acid, myristic acid, sodium palmitate, sodium stearate, and recombinant human albumin was set to 0.3:0.3:0.3:0.5:1, and the weight ratio of poloxamer to recombinant albumin was set to 10 μg to 500 μg of poloxamer / 1 g of recombinant albumin. Poloxamer was used to promote fatty acid dissolution. Step A involves injecting the sample, washing the anion exchange chromatography column with purified water to neutralize it (however, the chromatography column is packed to a height of 380 mm), eluting the recombinant human albumin component from the chromatography column adsorbed with the sample using a pH 6.5 eluent system of 50 mM phosphate buffer, and collecting the recombinant human albumin component. Step B includes the following steps: cation exchange chromatography equilibrates the chromatography column with a 40 mM acetate-NaOH equilibrium liquid system, desalts the collected recombinant human albumin components with a 10 kDa flat membrane and replaces them with the cation exchange chromatography equilibrium liquid system, then performs sample injection and chromatographic separation to collect the recombinant human albumin components; A method for producing high-purity and highly stable proteins, characterized by the above.

2. Step C involves replacing the recombinant human albumin sample collected by the previous purification with a 40 mM acetate-NaOH equilibrium liquid system in a 30 kDa membrane module, equilibrating the cation exchange chromatography column with the equilibrium solution, adding the mixture to the sample, making the mixture a mixture of fatty acid mixture and poloxamer, with a molar ratio of oleic acid, myristic acid, sodium palmitate, sodium stearate, and recombinant human albumin of 0.3:0.3:0.3:0.5:1, followed by sample injection and chromatographic separation to collect the recombinant human albumin components. Step D includes replacing the recombinant human albumin components collected in Step C with the equilibrium liquid system of Step A using a 10 kDa membrane module, equilibrium the anion exchange chromatography column with the equilibrium solution of Step A, and collecting the recombinant human albumin components by injecting and purifying the sample in the same manner as in Step A. A method for producing high-purity and highly stable proteins according to claim 1.

3. The further step, after step B and / or step D, is to perform fractionation of polymer aggregates and removal of small molecule substances using 100 kDa and / or 30 kDa and / or 10 kDa membrane modules when the chromatography is finally complete, and then replace and concentrate into a recombinant human albumin stock solution with a concentration greater than 20%. A method for producing high-purity and highly stable proteins according to claim 1 or 2.

4. The pH of the equilibrium solution under cation exchange chromatography conditions is 4.0 to 6.5, and the electrical conductivity under cation exchange chromatography conditions is 10 mS / cm or less. A method for producing high-purity and highly stable proteins according to claim 1.

5. The recombinant human albumin samples collected through the initial purification process are human albumin expressed in genetically modified microorganisms produced on a large scale. A method for producing high-purity and highly stable proteins according to claim 1.

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