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• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P7/00—Drugs for disorders of the blood or the extracellular fluid
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/76—Albumins
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
  • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
  • C07K14/76—Albumins
  • C07K14/765—Serum albumin, e.g. HSA
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07K—PEPTIDES
  • C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
  • C07K1/14—Extraction; Separation; Purification
  • C07K1/16—Extraction; Separation; Purification by chromatography
  • C07K1/18—Ion-exchange chromatography

Definitions

• the present invention provides a method for obtaining high-purity recombinant human albumin by adding medium- and long-chain fatty acid ligands and removing charge heterogeneity through anion and/or cation chromatography.
• human albumin The main pharmacological effects of human albumin include: regulating the dynamic balance of water between tissues and blood vessels, maintaining normal and constant plasma volume; at the same time, it has a high affinity for certain ions and compounds, and reversibly binds to these substances to exert a transport function; human albumin also provides the body with a large amount of amino acid reserves.
• Human albumin Due to the above-mentioned effects of human albumin, it can be applied to various clinical disciplines and exert a variety of therapeutic effects.
• Human albumin is mainly used clinically to regulate plasma colloidal osmotic pressure, expand blood volume, treat traumatic and hemorrhagic shock, severe burns and hypoproteinemia; it is also widely used in common diseases such as stroke, liver cirrhosis ascites and kidney disease.
• albumin has also been widely used in culture media used in vaccine production, pharmaceutical excipients, diagnostic reagents, new tumor long-acting preparations, cosmetics, laboratory biological reagents and many other aspects.
• human albumin is a single-chain non-glycosylated protein with a heart-shaped structure, 585 amino acids, 17 pairs of disulfide bonds, a free thiol group, and a molecular weight of 66438 Daltons.
• the half-life of human albumin in the human body is 19 to 21 days.
• the heart-shaped structure of human albumin consists of three main domains and six subdomains wrapped by 17 disulfide bonds, which are loosely bound together by van der Waals forces. From its crystal structure, it can be seen that the disulfide bridge gives the helical globular structure rigidity, but provides sufficient flexibility to enable the protein to undergo conformational changes according to changes in the surrounding medium.
• human albumin The conventional production method of human albumin is to extract, separate and purify it from human serum, which is collectively referred to as human serum albumin.
• Human albumin derived from human blood is subject to the quantity limitation of plasma sources, virus contamination of plasma donors, and individual antibody and protein differences, and there will be greater risks in its clinical use. Therefore, in many countries, there are virus safety statements in the instructions for use of human albumin, for example: "Standard measures taken to prevent infection caused by the use of human blood or plasma products include donor selection, screening of single blood donations or screening of plasma pools for special infection markers, and the use of effective production steps to inactivate/remove viruses. Even so, when using medicinal products prepared from blood or plasma, the possibility of infection by infectious pathogens cannot be ruled out. This includes unknown or emerging viruses and other pathogens.” Therefore, the use of genetic recombination is the best way to effectively obtain albumin without virus contamination.
• the most commonly used method for expressing human albumin by genetically recombinant microorganisms that can achieve large-scale production is mainly the yeast expression system, among which Saccharomyces cerevisiae and Pichia pastoris are the most mature.
• human albumin is a large-volume injection, the dose of each injection can reach 5 grams to 30 grams. Therefore, it is necessary to require that the total host protein residue and pollutants in the production process of each injection dose should not exceed 1ng/ml (200mg/ml-rHA).
• the recombinant albumin modified by glycosylation, oxidation, polymerization, mismatching and aggregation may produce immunotoxicity to the human body and may also have a certain impact on the stability of the protein. Therefore, an efficient and specific purification method is a key process element for obtaining high-purity homogeneous and uniform recombinant human albumin.
• Patent WO1966/037515 discloses a method of adding medium- and short-chain fatty acids such as sodium octanoate before ion exchange chromatography, which can remove glycosylated and heterogeneous albumin to a certain extent.
• sodium octanoate has a weak ligand capacity and cannot effectively remove mismatched, oxidized and truncated heterogeneous albumin.
• Patent CN2019108743433 discloses a method of adding medium-chain fatty acids or salts and a trace amount of polymer excipients to recombinant albumin preparations to improve the stability of albumin preparations and prevent agglomeration and aggregation.
• this method cannot selectively remove hydrophobic heterogeneity and charge heterogeneity, defective, isomerized or post-modified albumin, and cannot remove a small amount of unstable heterogeneous albumin from the source.
• the present invention adds a certain amount of medium-chain and long-chain fatty acids and fatty acid salts before refined ion exchange chromatography to meet the reasonable ligand ratio of albumin, assisting the effective spatial folding of albumin to show the charge heterogeneous albumin charge heterogeneous space.
• ion exchange chromatography can be used to effectively remove fragments and mismatches generated during fermentation and purification of recombinant albumin, as well as incorrect or post-modified albumin structures.
• the term “recombinant human albumin” may also be referred to as “recombinant albumin” and/or “recombinant human serum albumin” and/or “recombinant human albumin” and/or “rHA” and/or “rHSA”.
• human serum albumin refers to human albumin extracted from human serum, and may also be referred to as “human albumin” and/or “HSA” and/or “HA” and/or “pdHSA”.
• the term: “medium-chain fatty acids” refers to natural fatty acids with a carbon chain greater than 10 and their salts.
• Purpose of the invention To provide a better method for preparing high-purity and high-stability proteins.
• the specific purpose can be seen in the multiple substantial technical effects in the specific implementation part.
• a method for preparing a high-purity and high-stability protein characterized in that:
• Step A The recombinant human albumin sample collected by the previous purification was replaced with a buffer system of 80mM PB pH 7.5 using a Millipore 10KDa membrane package, and the sample collection solution was added into a mixed solution, wherein the mixed solution was a mixture of a fatty acid mixture and poloxamer; the molar ratio of oleic acid, myristic acid, sodium palmitate, sodium stearate and recombinant human albumin was 0.3:0.3:0.3:0.5:1;
• the anion exchange chromatography column was rinsed with purified water until it was neutral.
• the column was packed at a height of 380 mm.
• the column with the adsorbed sample was eluted with a 50 mM PB pH 6.5 eluent system to collect the recombinant human albumin component.
• Step B Use 40mM HAc-NaOH equilibrium liquid system to balance the chromatographic column for cation exchange chromatography, and collect the recombinant human albumin components; use Millipore 10KDa flat membrane desalting liquid to exchange for the cation exchange chromatography equilibrium liquid system, and then perform sample chromatography separation to collect the recombinant human albumin components.
• a method for preparing a high-purity and high-stability protein characterized in that:
• Step C The recombinant human albumin sample collected by the previous purification is replaced by a Millipore 10KDa membrane package with a 40mM HAc-NaOH equilibrium solution system as the buffer, and the cation exchange chromatography column is equilibrated with the equilibrium solution.
• a mixed solution is added to the sample, wherein the mixed solution is a mixture of a fatty acid mixture and poloxamer; 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; then, the sample is subjected to chromatographic separation to collect the components of the recombinant human albumin;
• Step D The recombinant human albumin component collected in the above step C is replaced with the buffer system of step A using a Millipore 10KDa membrane package, and the balance solution of step A is used to balance the anion exchange chromatography column. The same method is used to perform the sample purification of step A to collect the recombinant human albumin component.
• a further technical solution of the present invention is that after step B and/or step D, the following operation is also included: when the above-mentioned chromatography is finally completed, a 100KDa and/or 30KDa and/or 10KDa membrane package is used to intercept large molecular aggregates and remove small molecular substances, and replace and concentrate them into a recombinant human albumin stock solution with a concentration greater than 20%.
• the cation exchange medium includes but is not limited to SP strong cation exchange medium or CM weak cation exchange medium, and the cation exchange medium can be selected from 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 Boglund Biotech Co., Ltd.'s Bestarose series; anion exchange media include but are not limited to Q strong anion or DEAE weak anion exchange media, and anion exchange media may be selected from but are 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 Boglund Biotech Co., Ltd.'s Bestarose series.
• anion exchange media include but are not limited to Q strong anion or DEAE weak anion exchange media
• a further technical solution of the present invention is that the weight ratio of poloxamer to recombinant albumin is 10-500 micrograms of poloxamer per 1 gram of recombinant albumin; and poloxamer is used to promote the dissolution of fatty acids.
• a further technical solution of the present invention is that the anion exchange chromatography is purified by forward elution, wherein the pH of the equilibrium solution and the eluent are both between 6.0 and 9.5, and the conductivity under the conditions of the anion exchange chromatography described in the present invention is not higher than 20 ms/cm; or the anion exchange chromatography is purified by reverse elution, wherein the pH of the equilibrium solution is between 4.0 and 6.0, and the equilibrium conductivity under the conditions of the anion exchange chromatography is not higher than 10 ms/cm.
• a further technical solution of the present invention is that the anion exchange chromatography is purified by forward elution, and the pH of the equilibration solution and the eluent are preferably between 6.5 and 9.0; the conductivity under the conditions of the anion exchange chromatography is preferably not higher than 15 ms/cm; the anion exchange chromatography is purified by reverse elution, and the pH of the equilibration solution is preferably between 4.0 and 5.5, and the equilibrium conductivity under the conditions of the anion exchange chromatography is preferably not higher than 6 ms/cm.
• a further technical solution of the present invention is that the pH of the equilibrium solution in the cation exchange chromatography condition is between 4.0 and 6.5; and the conductivity in the cation exchange chromatography condition is not higher than 10 ms/cm.
• a further technical solution of the present invention is that the pH of the equilibrium solution in the conditions of the preferred cation exchange chromatography is between 4.5 and 6.0; and the conductivity in the conditions of the preferred cation exchange chromatography is not higher than 5 ms/cm.
• a further technical solution of the present invention is that the recombinant human albumin sample collected through preliminary purification is human albumin expressed by genetically recombinant microorganisms produced on a large scale.
• the present invention adopting the above technical scheme has the following beneficial effects compared with the prior art:
• the first uniqueness 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; that is, the molar ratio of long-chain fatty acids and fatty acid salts to human recombinant albumin is between 0.1 and 3:1; sequential use of ion exchange chromatography can effectively remove truncated albumin, mismatched or modified albumin in recombinant albumin, which may have side effects on the human body in clinical practice. Use or affect the half-life and efficacy of the drug.
• the second unique feature of the present invention is that by adding medium- and long-chain fatty acids and fatty acid salts before the refined ion exchange chromatography, the effective fatty acid ligand ratio can be selectively retained through ion exchange, and the components with excessive or insufficient ligand ratios due to hydrophobic heterogeneity caused by albumin structural modification or folding errors can be removed through ion exchange, thereby obtaining a high-purity and high-stability human albumin sample.
• FIG1 is a comparison diagram of the charge heterogeneity detection spectrum of the sample before Example 1 and the charge heterogeneity detection spectrum of the sample after sequential purification in Example 1 and Example 2; the ordinate of FIG1 is the absorption value (mAU), and the abscissa is the time axis (min);
• Figure 2 Accelerated stability experiment, DLS (dynamic light scattering) detects the particle size distribution of the test sample at 0, 1 month, and 3 months; the ordinate of Figure 2 is the distribution percentage; the abscissa is the molecular diameter (nm);
• Figure 3 shows a long-term stability experiment.
• DLS dynamic light scattering
• the fermentation scale of the present invention is implemented on 10L, 20L, 3,000L and 10,0000L equipment, and the purification scale is implemented on the scale of column diameter 10cm, 45cm, and 120cm, respectively, and its linear amplification is good.
• This embodiment includes but is not limited to the above scale.
• the following examples can further understand the characteristics and advantages of the present invention in conjunction with the accompanying drawings, and do not limit the remaining contents explained in any way by the present invention.
• the recombinant human albumin sample collected by the previous purification was replaced with a Millipore 10KDa membrane package with a 80mM PB pH 7.5 equilibrium solution system as the buffer.
• a mixture of oleic acid, myristic acid, sodium palmitate, sodium stearate and poloxamer was added to the sample collection solution, and the final concentration of fatty acids was 0.3:0.3:0.3:0.5:1 molar ratio (oleic acid: myristic acid: sodium palmitate: sodium stearate: recombinant human albumin).
• the UniGel-DEAE chromatography column (column loading height 380mm) was rinsed to neutrality with purified water.
• the chromatography column adsorbed with the sample was eluted with a 50mM PB pH 6.5 eluent system to collect the recombinant human albumin component.
• the cation exchange chromatography used SP Bestarose FF filler (equivalent to GE SP Sepharose Fast Flow filler) from Boglund Biotech (Shanghai) Co., Ltd.
• the chromatographic column was balanced with a 40mM HAc-NaOH balance liquid system, and the recombinant human albumin solution collected in Example 1 was desalted and replaced with a millipore 10KDa flat membrane as the balance liquid system, followed by sample chromatography separation to collect the components of the recombinant human albumin.
• Example 2 is first performed, and the recombinant human albumin sample collected by the previous purification is replaced with the buffer system of Example 2 using a Millipore 10KDa membrane package, and a mixture of oleic acid, myristic acid, sodium palmitate, sodium stearate and poloxamer is supplemented, and the final concentration of fatty acids is 0.3:0.3:0.3:0.5:1 molar ratio (oleic acid: myristic acid: sodium palmitate: sodium stearate: recombinant human albumin).
• the recombinant human albumin component is collected in the same manner as Example 2.
• Example 1 The collected recombinant human albumin components were replaced with the equilibrium solution system of Example 1 using a Millipore 30KDa membrane package and column equilibrium was performed. The same method was used to purify, load and collect the recombinant human albumin components as in Example 1.
• a 100KDa and/or 30KDa and/or 10KDa membrane package can be used to intercept macromolecular aggregates and remove small molecules, and to replace and concentrate into a recombinant human albumin stock solution with a concentration greater than 20%.
• Charge heterogeneity detection was performed on the sample before Example 1 and the sample after Example 1 and Example 2.
• the charge heterogeneity detection method refers to the ion chromatography method of the 2020 General Rules of the Fourth Volume of the Chinese Pharmacopoeia 0513 to determine the charge heterogeneity in recombinant human albumin.
• the test results are as follows ( Figure 1);
• This example is the protein solution purified by Example 2. After the stock solution production was finally completed, the recombinant human albumin injection was prepared; the injection sample was subjected to accelerated test under the conditions of temperature 25°C ⁇ 2°C and humidity 60% ⁇ 5%, generally observed for 6 months, test results (Table 1, Table 2), and attached figure ( Figure 1).
• This example is a protein solution purified by Example 2. After the stock solution production is finally completed, a recombinant human albumin injection is prepared. The injection sample is subjected to long-term stability at a temperature of 5°C ⁇ 3°C. Sexuality test, inspection test is generally: every three months in the first year, such as 0, 3, 6, 9, 12 months; every six months in the second year, such as 18, 24 months; and every year thereafter, such as 36 months. Test results (Table 3, Table 4), attached figure ( Figure 3);
• Table 2 Changes of Tonset , Tm1 and Tm2 of the test samples at 0 hours and 3 months after DSC testing.
• Figure 2 shows the particle size distribution of the test samples detected by DLS at 0 hours, 1 month, and 3 months. After the accelerated stability experiment, there was no significant change in the particle size distribution of the samples.
• Table 4 Changes of Tonset , Tm1 and Tm2 of the test samples detected by DSC at 0 hours and 36 months.
• Figure 3 DLS test particle size distribution of test samples at 0 hours, 12 months, 24 months, and 36 months. After long-term stability experiments, the particle size distribution of the samples did not show significant changes.
• the present invention provides a method for adding a certain amount of medium-chain fatty acids and fatty acid salts under the conditions of recombinant albumin purification intermediates, and removing the fragments and mismatched proteins with incorrect structures generated during the fermentation and purification of recombinant albumin by ion exchange chromatography.
• the method of the present invention purifies the purified recombinant human albumin solution by means of cation exchange chromatography, anion exchange chromatography, etc.
• the cation exchange medium of the present invention is an SP series, or a CM series cation exchange medium, which can be selected from but 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 Boglon Biotechnology Co., Ltd.'s Bestarose series.
• the medium of the anion exchange chromatography of the present invention is DEAE series, or Q series of strong anions.
• the anion exchange medium can be selected from but 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 Boglund Biotech's Bestarose series.
• Long chain fatty acids can be added before anion exchange chromatography or before cation exchange chromatography.
• the replacement buffer and concentration between the chromatography steps of the present invention can be implemented by using devices and equipment such as hollow fiber membranes and flat membrane packages with separation pore sizes between molecular weights of 1KDa and 30KDa, including but not limited to the order of use and cross-use.
• the replacement buffer between the chromatography steps of the present invention can also be processed by Sephadex G25 or Superdex G75.
• the recombinant albumin preparation with high charge consistency and hydrophobic consistency obtained after the implementation of the present invention can effectively meet the stability requirements of 50 hours at 57°C according to the prescription of the Chinese Pharmacopoeia (2020 edition) or the United States Pharmacopoeia (USP39), and can also be stably stored at 25°C or 30°C for more than 30 days.
• the poloxamer of the present invention is selected from one or more of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407.
• the method of the present invention obtains high-purity recombinant human albumin, which can be applied to the same clinical indications as serum albumin derived from human blood.
• the method of the invention obtains high-purity recombinant human albumin, which can be applied to diagnostic reagents, culture media and pharmaceutical excipients.
• the separation medium used in the present invention includes but is not limited to ion exchange media grafted with hydrophilic modified microspheres with a size of 10 ⁇ m to 150 ⁇ m composed of polyacrylate or polystyrene-divinylbenzene polymers, or Sepharose 6 FF, or any one of the above two polymer substrates or agarose matrix with a length of the grafted side chain.
• the column bed height used in the present invention is between 100 mm and 800 mm, preferably between 250 mm and 600 mm.
• the chromatographic conditions of the present invention can be adjusted and changed according to general instruction manuals.
• a 100KDa and/or 30KDa and/or 10KDa membrane package can be used to intercept macromolecular aggregates and remove small molecules, and replace and concentrate the recombinant human albumin stock solution with a concentration greater than the preparation concentration.
• the expression host cell described in the present invention is yeast, including Saccharomyces cerevisiae of the genus Saccharomyces, Kluyveromyces, Hansenula and Pichia.

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• 2022
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