Method for producing highly pure 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 provides a method for obtaining highly purified recombinant human albumin by adding a medium- to long-chain fatty acid ligand and removing charge heterogeneity by anion and / or cation chromatography. [Background technology]

[0002] The main pharmacological functions of human albumin include regulating the dynamic balance of water in tissues and blood vessels to maintain a normal and constant plasma volume; having a high affinity for certain ions and compounds and reversibly binding with these substances to perform transport functions; and storing a large amount of amino acids in the body.

[0003] Due to the above-mentioned functions of human albumin, it can be applied to various clinical fields to exert various therapeutic effects.Clinically, human albumin is mainly used to adjust the colloid osmotic pressure of plasma, expand blood volume, treat wound and hemorrhagic shock, severe burns and hypoproteinemia, and is also widely used in common diseases such as stroke, liver cirrhosis, hepatic ascites and kidney disease.In addition to its direct application in the field of clinical treatment, albumin is also widely used in multiple aspects such as culture media used in vaccine production, pharmaceutical adjuvants, diagnostic reagents, new long-acting tumor preparations, cosmetics, and experimental biological reagents.

[0004] Human albumin is a heart-shaped, single-chain, non-glycosylated protein with 585 amino acids, 17 disulfide bonds, one free thiol group, and a molecular weight of 66,438 daltons. Its half-life in the human body is 19-21 days. The heart-shaped structure of human albumin consists of three main domains and six subdomains bound together by 17 disulfide bonds, which are loosely held together by van der Waals forces. As seen in its crystal structure, the disulfide bridges provide rigidity to the helical globular structure, but also sufficient flexibility to allow the protein to undergo conformational changes in response to changes in the surrounding medium.

[0005] Human albumin is generally produced by extraction, separation, and purification from 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 limited quantities of plasma sources, viral contamination among plasma donors, and individual antibody and protein differences. Therefore, the instructions for use for human blood albumin in many countries all include a viral safety statement, stating, for example, "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 manufactured from blood or plasma, the possibility of infection by infectious agents cannot be excluded. This includes unknown or newly emerging viruses and other pathogens." Therefore, using recombinant DNA methods is the optimal route to effectively obtain albumin free of viral contamination.

[0006] Currently, the most common method for mass-producing recombinant human albumin through microbial expression is primarily yeast expression, with Saccharomyces cerevisiae and Pichia coli being the most mature. However, human albumin is a large-volume injectable drug, with each injection requiring a dose of 5–30 g. Therefore, the overall residual host protein content and contaminants present during the manufacturing process must be kept below 1 ng / ml (200 mg / ml rHA) for each injection. Regardless of the method used to produce recombinant human albumin, modifications such as glycosylation, oxidation, multimerization, mismatching, and aggregation can cause immunotoxicity in the human body and may also affect protein stability. Therefore, an efficient and specific purification method is a key element in the process of obtaining highly pure, homogenous, and uniform recombinant human albumin.

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

[0008] Patent CN2019108743433 discloses a method for adding medium- to long-chain fatty acids or salts and trace amounts of polymeric adjuvants to recombinant albumin preparations to improve the stability of the albumin preparations and prevent aggregation and aggregation. However, this method cannot selectively remove albumin with hydrophobic heterogeneity and charge heterogeneity defects, isomerism, or post-modification, and cannot remove small amounts of unstable heterogeneous albumin from the original.

[0009] The present invention adds a certain amount of medium- to long-chain fatty acids and fatty acid salts before purified ion exchange chromatography to achieve a reasonable ligand loading ratio for albumin, which helps albumin to effectively fold in space, resulting in charge-heterogeneous albumin and charge-heterogeneous space. Under these conditions, ion exchange chromatography can be used to effectively remove albumin with incorrect or post-modified structures, such as fragments and mismatches, generated during the fermentation and purification of recombinant albumin.

[0010] As used herein, 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." As used herein, the term "medium- to long-chain fatty acid" refers to naturally occurring fatty acids with a carbon chain of more than 10 and their salts. Summary of the Invention [Problem to be solved by the invention]

[0011] Object of the invention In order to provide a more effective method for producing a protein with high purity and high stability, the specific objectives are referred to in the specific embodiments, which have several substantial technical effects. [Means for solving the problem]

[0012] In order to achieve the above object, the technical means used in the present invention are as follows.

[0013] The method for producing a highly pure and highly stable protein is as follows: The purified and collected recombinant human albumin sample was replaced with a buffer system of 80 mM PB, pH 7.5, using a Millipore 10KDa membrane pack, and a mixture was added to the collected sample solution to form a mixture of fatty acid and poloxamer, with the molar ratio of oleic acid, myristic acid, sodium palmitate, sodium stearate, and recombinant human albumin being 0.3:0.3:0.3:0.5:1; Step A: after the sample is injected, the anion exchange chromatography column is washed with purified water until neutral, the chromatography column is filled to a height of 380 mm, and the chromatography column adsorbed with the sample is eluted with an elution liquid system of 50 mM PB, pH 6.5, to collect the recombinant human albumin component; The cation exchange chromatography method is characterized by including a step B in which the chromatography column is equilibrated with an equilibrium liquid system of 40 mM HAc-NaOH, and the collected recombinant human albumin components are desalted using a Millipore 10 KDa flat membrane and replaced with a cation exchange chromatography equilibrium liquid system, followed by sample injection and chromatographic separation, and the recombinant human albumin components are collected.

[0014] The method for producing a highly pure and highly stable protein is as follows: Step C: transferring the purified and collected recombinant human albumin sample to an equilibrium liquid system of 40 mM HAc-NaOH buffer using a Millipore 10KDa membrane pack, equilibrating a cation exchange chromatography column with the equilibrium liquid, adding a mixture to the sample, making the mixture a mixture of fatty acids 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, then injecting the sample and performing chromatographic separation to collect the recombinant human albumin components; and step D, in which the recombinant human albumin components collected in step C are substituted with the buffer system of step A using a Millipore 10KDa membrane pack, the anion exchange chromatography column is equilibrated with the equilibration solution of step A, and the sample is injected and purified in the same manner as in step A, and the recombinant human albumin components are collected.

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

[0016] In 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; the cation exchange medium may include, but is not limited to, 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 Bestarose series from BestChrom Biotechnology Co., Ltd.; the anion exchange medium includes, but is not limited to, Q strong anion or DEAE weak anion exchange medium; the anion exchange medium may include, but is not limited to, 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 Bestarose series from BestChrom Biotechnology Co., Ltd.

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

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

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

[0020] As a further technical means of the present invention, the pH of the equilibrium 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] In a further technical means of the present invention, the purified and collected recombinant human albumin sample is human albumin expressed in a genetically modified microorganism 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: The first unique feature of the present invention is that 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, i.e., the molar ratio of long-chain fatty acids and fatty acid salts to human recombinant albumin is 0.1-3:1, and ion exchange chromatography can be used subsequently to effectively remove cleaved albumin, mismatched or modified albumin in the recombinant albumin, which may clinically cause side effects in the human body or affect half-life and efficacy.

[0024] The second unique feature of the present invention is that by adding medium- to long-chain fatty acids and fatty acid salts before the purified ion exchange chromatography, the ratio of effective fatty acid ligands can be selectively retained by ion exchange, and components with too much or too little ligand ratio after hydrophobic heterogeneity occurs due to albumin structure modification or folding error can be removed by ion exchange, thereby obtaining a human albumin sample with high purity and high stability. [Brief explanation of the drawings]

[0025] To further explain the present invention, further reference is made to the following drawings.

[0026] [Figure 1] FIG. 1 is a comparative diagram of the charge heterogeneity detection spectrum of a sample before Example 1 and the charge heterogeneity detection spectrum of a sample after the purifications of Example 1 and Example 2 are performed in sequence. The vertical axis of FIG. 1 represents the absorption value (mAU), and the horizontal axis represents the time axis (min). [Figure 2] In the accelerated stability experiment, the particle size distribution of the measurement sample was detected by DLS (dynamic light scattering) at time 0, 1 month, and 3 months. The vertical axis of Figure 2 is the distribution percentage, and the horizontal axis is the molecular diameter (nm). [Figure 3]In a long-term stability experiment, the particle size distribution of the measurement sample was detected using a DLS (dynamic light scattering) at time 0, 12 months, 24 months, and 36 months. The vertical axis of Figure 3 is the distribution percentage, and the horizontal axis is the molecular diameter (nm). DETAILED DESCRIPTION OF THE INVENTION

[0027] The present invention will now be further described with reference to the drawings and specific embodiments, and it should be understood that the following specific embodiments are merely for the purpose of illustrating the present invention and are not intended to limit the scope of the present invention.

[0028] Fermentation scales of the present invention were carried out using 10 L, 20 L, 3,000 L, and 100,000 L facilities, and purification scales were carried out using column diameters of 10 cm, 45 cm, and 120 cm, respectively, and linear expansion was excellent. The present examples include, but are not limited to, the above scales. The following examples, taken in conjunction with the drawings, are intended to further understand the features and advantages of the present invention and are not intended to limit other aspects of the present invention in any way.

[0029] Example 1 Anion exchange chromatography The purified and collected recombinant human albumin sample was transferred to a Millipore 10KDa membrane pack with an equilibrated liquid system containing 80 mM PB, pH 7.5. A mixture of oleic acid, myristic acid, sodium palmitate, sodium stearate, and poloxamer was added to the sample collection liquid, resulting in a final fatty acid concentration of 0.3:0.3:0.3:0.5:1 molar ratio (oleic acid:myristic acid:sodium palmitate:sodium stearate:recombinant human albumin). After loading the sample, the UniGel-DEAE chromatography column (packed to a height of 380 mm) was flushed with purified water to neutral. The sample-adsorbed chromatography column was eluted with 50 mM PB, pH 6.5, and the recombinant human albumin component was collected.

[0030] Example 2 Cation exchange chromatography For cation exchange chromatography, SP Bestarose FF filler (equivalent to GE SP Sepharose Fast Flow filler) from BestChrom (Shanghai) Biotechnology Co., Ltd. was used. The chromatography column was equilibrated with a 40 mM HAc-NaOH equilibration liquid system, and the recombinant human albumin solution collected in Example 1 was desalted using a Millipore 10 KDa flat membrane and replaced with the equilibration liquid system. After that, the sample was injected and chromatographically separated to collect the recombinant human albumin components.

[0031] Example 3 Interchange between cation exchange chromatography and anion exchange chromatography In this example, the recombinant human albumin sample collected after the initial purification procedure described in Example 2 was first substituted with the buffer system described in Example 2 using a Millipore 10 KDa membrane pack, and a mixture of oleic acid, myristic acid, sodium palmitate, sodium stearate, and poloxamer was added to give a final fatty acid 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 in the same manner as in Example 2.

[0032] The collected recombinant human albumin component was replaced with the equilibration liquid system of Example 1 using a Millipore 30KDa membrane pack, column equilibration was performed, and purification, sample injection, and collection of the recombinant human albumin component were performed in the same manner as in Example 1.

[0033] Upon completion of the above chromatography, a 100 KDa and / or 30 KDa and / or 10 KDa membrane pack can be used to remove and replace the high molecular weight aggregate fraction and small molecular weight substances, resulting in a concentrated recombinant human albumin stock solution with a concentration of more than 20%. Charge heterogeneity was detected for the samples before and after Example 1 and Example 2.

[0034] The charge heterogeneity detection method was based on the ion chromatography method in the 2020 Fourth Part General Provisions of the Chinese Pharmacopoeia, 0513, to measure the charge heterogeneity in recombinant human albumin. The detection results were as follows (Figure 1):

[0035] Example 4 Acceleration stability experiment In this example, the protein solution purified in Example 2 was used to finally complete the production of the stock solution, and then recombinant human albumin injection was prepared. The injection samples were subjected to accelerated testing at a temperature of 25°C ± 2°C and a humidity of 60% ± 5%, and generally for up to 6 months. The test results (Tables 1 and 2) and Figure 1 are shown.

[0036] Example 5 Long-term stability experiments In this example, the protein solution purified in Example 2 was used to prepare a recombinant human albumin injection after final production of the original solution. The injection samples were then subjected to long-term stability testing at a temperature of 5°C ± 3°C. The stability was generally measured every three months in the first year (e.g., at 0, 3, 6, 9, and 12 months), every six months in the following year (e.g., at 18 and 24 months), and annually thereafter (e.g., at 36 months). The test results (Tables 3 and 4) are shown in Figure 3.

[0037] Table 1. Detection results [Table 1]

[0038] Table 2. T of samples measured by DSC at time 0 and 3 months onset , T m1 , T m2 A change in [Table 2]

[0039] The particle size distribution of the measured sample at time 0, 1 month, and 3 months was detected using DLS in Figure 2, and the accelerated stability experiment showed no significant changes in the particle size distribution of the sample.

[0040] Table 3. Detection results [Table 3]

[0041] Table 4. T of samples measured by DSC at time 0 and 36 months onset , T m1 , T m2 A change in [Table 4]

[0042] Figure 3: DLS was used to detect the particle size distribution of the measurement sample at time 0, 12 months, 24 months, and 36 months. The long-term stability experiment showed no significant changes in the particle size distribution of the sample.

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

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

[0045] The cation exchange medium of the present invention is an SP series, or a CM series cation exchange medium, which may include, but is not limited to, 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 Bestarose series from BestChrom Biotechnology Co., Ltd.

[0046] The anion exchange chromatography medium of the present invention is the DEAE series or the strong anion Q series, 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 Bestarose series from BestChrom Biotechnology Co., Ltd.

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

[0048] The buffer replacement and concentration during the chromatography step of the present invention can be performed using devices and equipment such as hollow fiber membranes and flat membrane packs with separation pore sizes ranging from 1 KDa to 30 KDa in molecular weight, including, but not limited to, sequential use and cross-use.

[0049] Buffer exchange during the chromatography steps of the present invention may be carried out using Sephadex G25 or Superdex G75.

[0050] The recombinant albumin preparation with high charge and hydrophobicity matching obtained after implementing the present invention can effectively meet the stability requirements of the Chinese Pharmacopoeia (2020 edition) or the United States Pharmacopoeia (USP39) when left for 50 hours at 57°C, and can also be stably stored for more than 30 days at 25°C or 30°C.

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

[0052] The highly pure recombinant human albumin obtained by the method of the present invention can be applied to the same clinical indications as serum albumin derived from human blood.

[0053] The highly purified recombinant human albumin obtained by the method of the present invention can be applied to diagnostic reagents, culture media and pharmaceutical adjuvants.

[0054] The separation media used in the present invention include, but are not limited to, ion exchange media of 10 μm to 150 μm hydrophilically modified grafted microspheres made of polyacrylate or polystyrene-divinylbenzene polymer, and may also be Sepharose 6 FF, or any one of the above two polymer substrates or agarose matrices with grafted side chain lengths.

[0055] In the present invention, the column bed is packed to a height of 100 mm to 800 mm, preferably 250 mm to 600 mm.

[0056] The chromatographic conditions described in this invention can be adjusted and modified to some extent according to general manuals.

[0057] During the chromatography described in this 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, a 100 KDa and / or 30 KDa and / or 10 KDa membrane pack can be used to fractionate macromolecular aggregates and remove small molecular substances, and then the resulting recombinant human albumin stock solution can be concentrated to a concentration greater than that of a pharmaceutical preparation.

[0059] Expression host cells according to the present invention are yeasts, including those of the genus Saccharomyces, Kluyveromyces, Hansenula and Pichia.

[0060] The basic principles, main features and advantages of the present invention have been described above. Those skilled in the art should understand that the present invention is not limited to the above embodiments, and that the above embodiments and descriptions in the specification are merely for illustrating the principles of the present invention, and that the present invention also has various changes and modifications without departing from the spirit and scope of the present invention, and all of these changes and modifications are 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. Anion exchange chromatography is a purification method using a binding elution method, where 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 anion exchange chromatography is a purification method using a non-adsorption recovery elution method, where 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. A method for producing high-purity and highly stable proteins according to claim 1.

5. 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.

6. 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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