RU2801099C1 - Medicinal product containing human albumin and a method of its production - Google Patents

Abstract

FIELD: medicine.

SUBSTANCE: group of inventions relates to a preparation containing human albumin. The medicinal product containing human albumin, the specified medicinal product contains poloxamer and fatty acids, while the molar ratio of fatty acids and human albumin in the specified preparation is 0.5–2.0:1.0, the content of poloxamer in the specified preparation is 10–100 µg poloxamer/1 g human albumin, where the said poloxamer is one or more poloxamers selected from poloxamer 188 and poloxamer 407, where the said fatty acid is one or more acids selected from myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0) and oleic acid (C18:1) wherein the human albumin is human serum albumin or human recombinant albumin. A method of producing a medicinal product containing human albumin, including the following: providing human albumin, adding 10–100 μg of poloxamer per 1 g of human albumin, and adding fatty acids to achieve a molar ratio of fatty acids to human albumin equal to 0.5–2.0:1.0, where the said poloxamer is one or more poloxamers selected from poloxamer 188 and poloxamer 407, where the said fatty acid is one or more acids selected from myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0); and oleic acid (C18:1) wherein the human albumin is human serum albumin or human recombinant albumin.

EFFECT: human albumin preparation described above has improved stability with a small amount of poloxamer.

9 cl, 11 dwg, 2 tbl

Description

Technical field

[0001] The present invention relates to the field of biomedicine and, in particular, to a medical product containing human albumin, and a method for its production. According to the present invention, the term "human albumin" includes human serum albumin and recombinant human albumin. Recombinant human albumin is also called recombinant human serum albumin or mutant recombinant human albumin. State of the art

[0002] The main pharmacological effects of human albumin include the regulation of the dynamic balance of water between tissues and blood vessels, the maintenance of normal and constant blood plasma volume, and the transport of specific ions and compounds by reversible binding to them due to the high affinity for these substances. Moreover, human albumin provides a large supply of amino acids for the body. The effects of human albumin mentioned above allow it to be used in various clinical areas for various therapeutic purposes. Human albumin is mainly used in clinical practice to regulate plasma colloid osmotic pressure, increase blood volume, treat traumatic and hemorrhagic shock, severe burns and hyperproteinemia, and is widely used to treat common diseases such as stroke, cirrhosis and hepatic ascites, as well as diseases kidneys. In addition to direct use in clinical therapy, albumin has been widely used in many products, such as culture media used in vaccine production, pharmaceutical excipients, diagnostic reagents, new long-acting tumor drugs, cosmetics, and laboratory bioreagents.

[0003] Human albumin is a single chain non-glycosylated protein with a heart-shaped structure and a molecular weight of 66.438 Da, consisting of 585 amino acids, 17 pairs of disulfide bridges and one free sulfhydryl group. Its half-life in humans is between 19 and 21 days. The heart-shaped structure of human albumin consists of three main domains, which are loosely linked together by van der Waals forces, and six subdomains, surrounded by 17 disulfide bridges, as seen from its crystal structure. The disulfide bridges stiffen the helical globular structure, but provide sufficient mobility to allow the protein to undergo conformational changes in response to environmental changes.

[0004] Human albumin is usually obtained by fractionation, extraction and purification from human serum and is generally referred to as human serum albumin. Human serum albumin has been discovered and used for many years, and complete and well-developed processes for its preparation and dosage form have been developed (Kohn method - ultrafiltration or purification - pasteurization - dosage form (addition of sodium caprylate and / or N-acetyl tryptophan )). Its dosage form has become the common standard for national pharmacopoeias around the world. Albumin, derived from human serum, is characterized by good stability. In accordance with pharmacopoeial requirements, the final product should be stored in a water bath at a temperature of 57°C ± 0.5°C for 50 hours, while no visual changes should be observed, except for a slight color change. All end products of serum-derived albumin should be stored at 20-25°C for at least 4 weeks or at 30-32°C for at least 14 days. Containers showing cloudiness or cloudiness should be subjected to appropriate investigations. Despite such a rigorous study of the thermostability of human serum albumin, phlebitis associated with microaggregates and proteinaceous fibrillar aggregates still occurs in clinical practice. Clinical studies have shown that the incidence of phlebitis in patients is 20% with conventional infusion systems, including the use of filters with a pore size of 15 μm for intravenous infusion of human serum albumin, but only 2.5% with targeted infusion systems using filters with pore size 5 µm. In this regard, it was demonstrated that after sterilization and removal of particles by filtering through a filter with a pore size of 0.1-0.22 μm in the process of obtaining human serum albumin, new microaggregates and microfibrillar proteins still appear in the medical product during transportation and storage. leading to a high percentage of albumin infusion-induced phlebitis in patients (Xiao-Ping Xue et al., Journal of Clinical Nursing, 2010 Oct, 9(5): 69-70). Thus, the requirements for targeted infusion systems are specified in the human serum albumin specifications of some manufacturers. However, the partially unstable albumin mentioned above has a diameter of 30-100 nm and therefore cannot be effectively removed with conventional sterilization membranes (0.1-0.22 µm). Aggregates with this diameter, which are between the sizes of polymers and visible particles, are the main cause of adverse reactions such as immunogenicity.

[0005] Another source of human albumin is recombinant human albumin, which is expressed in a host capable of specifically expressing human albumin, such as Pichia pastoris or Saccharomyces cerevisiae - unicellular fungal microorganisms cultivated using genetic recombination techniques, or recombinant human albumin expressed transgenic rice. However, the highly purified end product of recombinant human albumin, which meets the requirements for formulations specified in pharmacopoeias, is much less stable compared to albumin derived from human serum. In view of this shortcoming, the Chinese Patent Application (CN Application No. 201610017874.7) described a method for preventing the formation of recombinant human albumin aggregates by adding Tween 80 or Tween 20 to a pharmacopeia standard dosage form.

[0006] S. Baldursdottir et al. the stabilizing effect of the excipient polysorbate 80 (Tween 80) in solutions of recombinant human albumin has been described. The excipients Caprylate and Polysorbate 80 used in the Recombumin Alpha® formulation have been reported to result in increased protein stability. Caprylate appeared to be effective in protecting the protein at higher temperatures, while polysorbate (Tween) protected the protein from self-association at lower temperatures. In addition, a non-linear synergistic effect of caprylate and polysorbate 80 was observed (S. Baldursdottir, M. Tauhaybeche, J. Pajander, J.T. Bukrinski, L. Jorgensen, Screening of formulation parameters for stabilizing recombinant human serum albumin (rHSA) in liquid formulations , Journal of Drug Delivery Science and Technology (2016), doi: 10.1016/j.jddst.2016.05.001.)

[0007] The negative effect of polysorbate is to increase the likelihood of protein oxidation catalyzed by residual alkyl oxides in the nonionic surfactant. For example, recombinant human ciliary neurotrophic factor (CNTF) can be dimerized by alkyl peroxide from polysorbate 80. The content of alkyl peroxides in polysorbate 80 correlates with the level of oxidation of recombinant human granulocyte colony stimulating factor (r hG-CSF), and peroxide mediated oxidation appears to be more strong compared to oxidation mediated by atmospheric oxygen present in the air space of the ampoule (Knepp VM, Whatley JL, Muchnik A, Calderwood TS. 1996. Identification of antioxidants for prevention of peroxide-mediated oxidation of recombinant human ciliary neurotrophic factor and recombinant human nerve growth factor PDA J Pharm Sci Technol 50(3): 163-171 Herman AC, Boone TC, Lu HS 1996 Characterization, preparation, and stability of Neupogen (Filgrastim), a recombinant human granulocyte-colony stimulating factor Pharm Biotechnol 9:303-328).

[0008] Human albumin contains methionine and a free sulfhydryl group to protect cells and organs from free radical peroxidation damage. Thus, the quality and storage conditions of polysorbate must be well regulated and its relative amount in proteins must be kept to a minimum (AGGREGATION OF THERAPEUTIC PROTEINS, P341, Edited by Wei Wang, Christopher J. Roberts, WILEY 2010), otherwise human albumin will be partially oxidized, losing the corresponding redox physiological activity; while oxidized human albumin will contribute to the additional unfolding of human albumin, accelerating its aggregation and fibrillation.

[0009] One of the very important physiological functions of human albumin in the bloodstream is the transport of drugs, fatty acids, vitamins, cytochromes and metal ions, based on its ability to bind ligands. [0010] Fatty acids, as ligands for human albumin, play a critical role in the structure and thermal stability of human albumin. The addition of sodium caprylate to human serum albumin as a ligand stabilizer can effectively increase the enthalpy value and Tm (melting point), thus enhancing thermal stability.

[0010] Brian et al. described the use of differential scanning calorimetry (DSC) to determine the unfolding capacity of commercial human albumin products derived from a pool of human blood, transgenic yeast, and transgenic rice. After the addition of oleic acid, the initial values of the melting point (Tm1) for conformational transitions with the unfolding of human albumin products were variously increased from 62°C to 75°C. This study showed the importance of fatty acid ligands for the thermal stability of human albumin, however, aggregation and fibrillation caused by exposure of the hydrophobic region of the protein was not investigated, and DSC was unable to characterize aggregation and fibrillation at low temperature and ambient temperatures, as well as under conditions fluctuations during transport (Brian E. Lang and Kenneth D. Cole, Biotechnol. Prog., 2014, Unfolding Properties of Recombinant Human Serum Albumin Products Are Due To Bioprocessing Steps, DOI 10.1002/btpr.l996).

[0012] In addition to fatty acids, there are additional ligands such as bilirubin, hormones, fat-soluble drugs, and those that bind strongly to human serum albumin, increasing the conformational stability of albumin. Defatted human serum albumin has a significantly reduced stability, which is fully consistent with the data for highly purified recombinant human albumin.

[0013] Surfactants such as Tween, when used as stabilizers, should be added in the smallest possible amount to avoid their toxic effects and, more importantly, to suppress the overlapping effects of Tween and other surfactants with physiological properties. human albumin ligands. Otherwise, safer surfactants with lower doses should be sought.

[0014] Poloxamer is the generic name for copolymers of polyoxyethylene and polyoxypropylene. Poloxamers, manufactured by BASF in Germany and known under the trade name Pluronic, are non-ionic surfactants. Poloxamers are exclusively synthetic emulsifiers currently used in intravenous emulsions, of which poloxamer 188 has the best emulsifying power and safety, being an oil-in-water emulsifier that is typically used at a concentration of 0.3% (Wang Meng et al., Pharmaceutical and Clinical Research 2007, 15(1): 10-13).

[0015] As a non-ionic surfactant, poloxamer 188 can increase the solubility of insoluble drugs by forming micelles at a critical micelle concentration (CMC) of about 0.6%. Poloxamer 188 can inhibit thrombosis, affect blood rheology, thicken cell membranes, phagocyte activation (stimulated phagocytosis and production of supernegative ions) and neutrophil degranulation, and prevent skeletal muscle necrosis in adults. In addition, poloxamer 188 can be used to treat brain damage. Studies have shown that poloxamer 188 can reduce inflammation and tissue damage in rats caused by experimental brain injury. Moreover, data from other experimental models have shown that it counteracts tissue damage. Its defense mechanism may relate to the effects of the surfactant on oxidative stress and the inflammatory response. Moreover, poloxamer 188 can enhance cell survival. Studies have shown that a single intravenous dose of poloxamer 188 is effective in repairing damaged tissue in an intact circulation. Poloxamer 188 is able to stabilize defects in cell membranes caused by various reasons. Studies in rats have shown that a single dose of poloxamer 188 counteracts early neuronal damage after bleeding, but is ineffective in neuronal damage caused by prolonged bleeding. However, daily administration of poloxamer 188 can provide long-lasting and effective neuronal protection.

[0016] Chinese Patent Application No. CN 200480003079.8 describes the use of poloxamer as a surfactant to stabilize hematopoietic factors, parathyroid hormones or antibodies. In this patent, no ligand effect on proteins was observed for fatty acids, so a higher dose of poloxamer was added to stabilize proteins and avoid Tween-mediated oxidative damage. Since hematopoietic factors, parathyroid hormones or antibodies are contained in injection preparations in a small volume (µg-mg/ampoule), the total content of poloxamer is negligible even if higher concentrations are added. However, human albumin is contained in the injectable preparations in high volume with an injection dose of 5-12.5 g/ampoule, thus the total content of Tween or poloxamer is higher even if they are added at a low concentration.

[0017] Thus, those skilled in the art are interested in researching a medical product containing human albumin, or a method for producing it, to suppress the formation of aggregates and fibrillar microparticles of human albumin and to improve the safety and tolerability of human albumin in clinical use with the prevention of toxicity mediated by Tween, and oxidative denaturation of proteins caused by Tween oxides present in the dosage forms described in the publications.

BRIEF DESCRIPTION OF THE INVENTION

[0018] The technical problem to which the present invention is directed is to improve the stability of human albumin preparations.

[0019] To solve the stated technical problem, the present invention proposes a method for obtaining a medical product containing human albumin, including: increasing the stability of human albumin in the medical product by adjusting the content of long chain fatty acids in the specified medical product and adding poloxamer to the specified medical product.

[0020] Preferably, the method further comprises: improving the stability of human albumin in the medical product by reducing aggregation and fibrillation of human albumin.

[0021] Preferably, the method further comprises: improving the stability of human albumin in the medical product so that particles larger than 30 nm in diameter are not formed due to aggregation and/or fibrillation of human albumin after the medical product is prepared.

[0022] Preferably, the method further comprises: improving the stability of human albumin in the medical product so that particles larger than 100 nm in diameter do not form as a result of aggregation and/or fibrillation of human albumin after formulation.

[0023] Preferably, the method further comprises: improving the stability of human albumin in the medical product so that particles larger than 200 nm in diameter do not form as a result of aggregation and/or fibrillation of human albumin after preparation of the drug.

[0024] Preferably, the method further comprises: improving the stability of human albumin in the medical product so that the stability meets the requirements in accordance with national pharmacopoeias.

[0025] Preferably, the method further comprises: increasing the stability of human albumin in the medical product so that the stability satisfies the following conditions: no haze or formation of visible particles during incubation at 57°C for 50 hours, incubation at 30°C for 14 days and incubation at 20°C for 28 days.

[0026] Preferably, the human albumin is human serum albumin, and said method includes: improving the stability of human albumin in a medical product by adding a poloxamer.

[0027] Preferably, the human albumin is recombinant human albumin, and said method includes: improving the stability of human albumin in a medical product by adding long chain fatty acids and a poloxamer.

[0028] Preferably, the long chain acid is added as a salt.

[0029] Preferably, the long chain acid is added as the sodium salt.

[0030] Preferably, the method further includes the following condition: after obtaining a medical product, the molar ratio of long chain fatty acids and human albumin is 0.2-3.0.

[0031] Preferably, the method further includes the following condition: after obtaining a medical product, the molar ratio of long chain fatty acids and human albumin is 0.3-2.0.

[0032] Preferably, the method further includes the following condition: after obtaining a medical product, the content of poloxamer is 10-500 μg/g of human albumin.

[0033] Preferably, the poloxamer is one or more poloxamers selected from poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407.

[0034] Preferably, the poloxamer is poloxamer 188.

[0035] Preferably, the long chain fatty acid is one or more selected from myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acids (C18:2), linolenic acid (C18:3) and arachidonic acid (C20:4).

[0036] Preferably, the long chain fatty acid is a soy fatty acid or a fully hydrogenated soy fatty acid.

[0037] Preferably, the method further comprises: adding ingredients in amounts according to national pharmacopoeias.

[0038] Preferably, the ingredients required by national pharmacopoeias include sodium caprylate and/or N-acetyltryptophan.

[0039] The present invention further provides a medical product containing human albumin obtained in accordance with the method of the present invention.

[0040] The present invention further provides a medical product containing human albumin that does not contain particles larger than 30 nm in diameter resulting from aggregation and/or fibrillation of human albumin.

[0041] According to the present invention, there is further provided a medical product containing human albumin, which does not contain particles with a diameter of more than 100 nm, formed as a result of aggregation and/or fibrillation of human albumin.

[0042] According to the present invention further provided a medical product containing human albumin, which does not contain particles with a diameter of about 200 nm, formed as a result of aggregation and/or fibrillation of human albumin.

[0043] The present invention further provides a medical product containing human albumin, wherein said method further comprises the following condition: the stability of human albumin in the medical product satisfies the following requirements: no haze or formation of visible particles during incubation at 57°C for 50 hours , incubation at 30°C for 14 days and incubation at 20°C for 28 days.

[0044] The present invention further provides a medical product containing human albumin that contains poloxamer and long chain fatty acids.

[0045] Preferably, the molar ratio of long chain fatty acids and human albumin in the medical product is 0.2-3.0.

[0046] Preferably, the molar ratio of long chain fatty acids and human albumin in the medical product is 0.3-2.0.

[0047] Preferably, the method further includes the following condition: the content of poloxamer in the medical product is 10-500 μg/g of human albumin.

[0048] Preferably, the poloxamer is one or more poloxamers selected from poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407.

[0049] Preferably, the poloxamer is poloxamer 188.

[0050] The long chain fatty acid is preferably one or more selected from myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acids (C18:2), linolenic acid (C18:3) and arachidonic acid (C20:4).

[0051] The long chain fatty acid is preferably a soy fatty acid or a fully hydrogenated soy fatty acid.

[0052] Preferably, the medical product further contains ingredients in amounts according to national pharmacopoeias.

[0053] Preferably, said ingredients required by national pharmacopoeias include sodium caprylate and/or N-acetyltryptophan.

[0054] The present invention is directed to improving the stability of human albumin based on the reduction of thermal denaturation by the use of fatty acid ligands, where the stability is improved by reducing aggregation and fibrillation at low temperature, ambient temperature and high temperature, or during fluctuations during transportation to based on the use of poloxamer as hydrophobic patches screening hydrophobic interactions and intersurface hydrophobic interactions. The diameter of such aggregates and fibrillar particles is 30-300 nm, mainly between 100 and 200 nm, which makes it impossible to detect them using size exclusion HPLC analysis, and also to filter them effectively using membrane systems with a pore size of 0.1-0 .2 µm and, more importantly, such aggregates continuously form in unstable albumin solution systems (even if aggregates and fibrillar particles have been completely removed) during storage and transport and continue to grow to form visible particles having a diameter of the micron and submillimeter range.

[0055] In order to ensure the suppression of aggregation and fibrillation of human albumin under various conditions, the authors of the present invention conducted extremely detailed studies for the analysis of albumin from human blood plasma. It has been found that 0.3 to 0.8 moles of long chain fatty acids are found in each mole of human plasma albumin. At the same time, 0.5 to 2.5 mol of long-chain fatty acids are contained in each mol of human albumin circulating in the human body to ensure the physiological functions of fatty acid mobilization and storage. Some of the long chain fatty acids are lost during the purification process during the production of albumin from the human blood pool. According to 2015 Chinese Pharmacopoeia Volume III, 0.140-0.180 mmol of sodium caprylate is added to the pasteurized medical product per 1 g of albumin; or use sodium caprylate together with acetyltryptophan, while adding 0.064-0.096 mmol of sodium caprylate and 0.064-0.096 mmol of acetyltryptophan per 1 g of albumin. At the same time, being a drug for injection in a large volume, the medical product must comply with the physiological conditions of the human body. The pH should be 6.4-7.4 and the osmolality should be 210-400 mOsm/kg.

[0056] Regardless of how recombinant human albumin is expressed, a multi-step purification process, including fractionation, chromatography, ultrafiltration and dialysis, etc., is required to completely remove proteins, polysaccharides, endotoxins and various impurities from the host body, at the same time, fatty acid ligands are also completely removed, resulting in defatted human albumin. Thus, the stability of recombinant human albumin that has lost fatty acids or various other ligands present in the human body is significantly lower than that of albumin derived from human plasma. Thus, some surfactants must be added below the CMC to act as a hydrophobic patch for albumin in the absence of ligands to prevent post-unfolding aggregation from interactions at various hydrophobic interfaces such as albumin/air/container/bubbles. air generated as a result of vibrations, etc.

[0057] Thus, based on the General Requirements for Preparations and Human Serum Albumin according to the Chinese Pharmacopoeia, American Pharmacopoeia, European Pharmacopoeia and other pharmacopoeias, a certain amount of poloxamer is added to the product and the ratio of long chain fatty acids is adjusted according to the present invention in such a way as to ensure compliance requirements for the stability of human albumin under the conditions specified in each national pharmacopoeia.

[0058] The advantage of the present invention is that the direct addition of long chain fatty acid ligands at a ratio characteristic of human blood flow along with a small amount of poloxamer, or the use of existing standard dosage forms of human albumin with the addition of an additional small amount of poloxamer leads to thermal stability of human albumin and the absence of aggregation and fibrillation during storage and transportation. The diameter of such aggregates and fibrillar particles is 50-300 nm, mainly between 100-200 nm, which does not allow them to be detected by analysis using size exclusion HPLC, and also effectively filtered using membrane systems with a pore size of 0.1-0 .2 µm and, more importantly, such aggregates are constantly formed in unstable albumin solution systems (even if the aggregates have been completely removed) during storage and transport and continue to grow to form visible particles with a diameter of the micron and submillimeter range.

[0059] The idea, specific structure, and resulting technical effects of the present invention will be further described hereinafter along with accompanying drawings in order to provide a thorough understanding of the purpose, features, and effects of the present invention.

BRIEF DESCRIPTION OF GRAPHICS

[0060] FIG. 1 is a dynamic light scattering chromatography (DLS) chromatogram of sample 1 (human serum albumin containing sodium caprylate and acetyl tryptophan) according to embodiment 1.

[0061] FIG. 2 is a DLS chromatogram of sample 2 (human serum albumin containing sodium caprylate and acetyltryptophan with poloxamer added) according to embodiment 1.

[0062] FIG. 3 is a DLS chromatogram of sample 3 (commercial human serum albumin containing only sodium caprylate) according to embodiment 2.

[0063] FIG. 4 is a DLS chromatogram of sample 4 (commercial human serum albumin containing only sodium caprylate with the addition of poloxamer) according to embodiment 2.

[0064] FIG. 5 is a DLS chromatogram of sample 5 (recombinant human albumin containing sodium caprylate and acetyltryptophan without the addition of poloxamer or long chain fatty acids) according to embodiment 3.

[0065] FIG. 6 is a DLS chromatogram of sample 6 (recombinant human serum albumin containing sodium caprylate and acetyltryptophan with the addition of only a long chain fatty acid (oleic acid)) according to embodiment 4.

[0066] FIG. 7 is a DLS chromatogram of sample 7 (recombinant human albumin containing sodium caprylate and acetyltryptophan with the addition of poloxamer only) according to embodiment 5.

[0067] FIG. 8 is a DLS chromatogram of sample 8 (recombinant human albumin containing sodium caprylate and acetyltryptophan supplemented with poloxamer and a long chain fatty acid (oleic acid)) according to embodiment 6.

[0068] FIG. 9 is a DLS chromatogram of sample 9 (recombinant human albumin containing sodium caprylate and acetyltryptophan supplemented with poloxamer and long chain fatty acid (sodium soyates)) according to embodiment 7.

[0069] FIG. 10 is a DLS chromatogram of sample 10 (recombinant human albumin containing sodium caprylate and acetyltryptophan with the addition of poloxamer and long chain fatty acids (a mixture of sodium stearate and sodium oleate)) according to embodiment 8.

[0070] FIG. 11 is a DLS chromatogram of sample 11 (recombinant human albumin containing sodium caprylate with poloxamer and long chain fatty acids (mixture of sodium stearate and sodium oleate)) according to embodiment 9.

DESCRIPTION OF VARIANTS FOR IMPLEMENTING THE INVENTION

[0071] Embodiments of the present invention are illustrated below with specific examples. Other advantages and effects of the present invention will be well understood by a person skilled in the art based on the description given in the application. The invention may also be carried out or applied in accordance with various other embodiments. The details given in this application can also be modified or changed in various ways in accordance with various intentions and applications within the essence of the present invention. It should be noted that the following embodiments of the invention and features of these embodiments can be combined with each other in the absence of contradictions.

[0072] The following are definitions and explanations of terms used in this application.

[0073] The term "human albumin" means human serum albumin or recombinant human albumin, where said recombinant human albumin is also referred to as recombinant human serum albumin or mutant recombinant human albumin. Recombinant human albumin includes at least human albumin derived from a genetically recombinant microorganism, recombinant human albumin from a eukaryotic cell, recombinant human albumin from a transgenic plant, and recombinant human albumin from a transgenic animal, and so on.

[0074] The term "medical product" should be understood to include at least a solution product, a freeze-dry product, or a low-temperature spray-dry product, preferably a solution product that, usually placed under aseptic conditions in a glass or plastic container of a certain volume.

[0075] It should be understood that the term "medical product containing human albumin" means medical products for clinical injection, culture media, drug excipients, medical devices, vaccine excipients, cosmetic products, analytical diagnostic reagents, and many other products containing human albumin at various concentrations, for example at a concentration of 1%-30%, preferably 10%-25%.

[0076] Poloxamer is a non-ionic surfactant block copolymer of ethylene oxide and 1,2-epoxypropane with the general formula H(C2H4O) a(C3H6O) b(C2H4O) aOH. Poloxamer 188 is highly biosafe and is listed as an emulsifier for intravenous fat emulsions and stabilizers for antibody injection according to the Chinese Pharmacopeia 2015 edition, USP and European Pharmacopoeia and for use in approved medicinal products.

[0077] It should be understood that the term "long chain fatty acids" refers to fatty acids containing more than 12 carbon atoms in the hydrocarbon chain, and means fatty acids of various origins (synthetic or isolated) that are acceptable to humans, or fatty acids that are present in various forms (for example, salts, in particular sodium salts), including, but not limited to, one or more of myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3) and arachidonic acid (C20:4).

[0078] The term "regulation" in relation to the content of long-chain fatty acids in a medical product containing human albumin, means maintaining the content of long-chain fatty acids in a medical product containing human albumin, within this range: in the case of long-chain fatty acids characteristic of containing human albumin of a medical product obtained by existing methods (such as a medical product containing human albumin originating from human blood), if the content of long chain fatty acids is below this range, "adjustment" means adding long chain fatty acids in the process of obtaining said medical product, containing human albumin, so that the amount of long-chain fatty acids in the final medical product containing human albumin is within this range; if the content of long-chain fatty acids is already within this range, "regulation" means that no action is required in the process of obtaining said medical product containing human albumin; if the medical product containing human albumin obtained using the existing method does not contain or contains a very small amount of long chain fatty acids, "adjustment" means the addition of long chain fatty acids in the process of obtaining said medical product containing human albumin, so that the content of the specified long chain fatty acids in the final medical product containing human albumin was within this range.

[0079] It should be understood that the term "long chain fatty acids" is an established phrase, where there is no logical relationship between the presence of the "O-R" substituent in the specified fatty acid and the content of the specified phrase. In the case of calculating the content of "long chain fatty acids", the term "long chain fatty acids" refers to the sum of the amounts of each long chain fatty acid. Any long chain acid or mixture of long chain fatty acids may be added to ensure that the content of long chain fatty acids is within this range.

[0080] It should be understood that the term "long chain fatty acid content" means the content of long chain fatty acids in the final medical product containing human albumin, including the amount of long chain fatty acids remaining during the preparation process due to the presence of long chain fatty acids characteristic of the source material, and the amount of additional added long chain fatty acids (if there is an addition in the process of "regulation").

[0081] It should be understood that the term "add" when used in relation to poloxamer, means adding poloxamer in the process of obtaining a medical product containing human albumin, so that the content of said poloxamer in the final medical product containing human albumin is within this range.

[0082] It should be understood that the steps of "adjusting" and "adding" are carried out in the process of obtaining a medical product containing human albumin, so that the content of long chain fatty acids and the content of poloxamer in the final medical product containing human albumin are within a given range. range. For example, these steps can be performed after the final bulk dosage form is obtained, or these steps can be performed during the ultrafiltration and dialysis processes, or poloxamer and long chain fatty acids can be added as a protein stabilizer before the pasteurization process, or these steps can be performed at the last one or several stages of the purification process using chromatography to obtain the range of values of the content of poloxamer and long chain fatty acids, specified according to the present invention, in the final medical product containing human albumin.

[0083] It should be understood that the term "improvement" means a higher stability of the medical product containing human albumin according to the present invention compared to the stability of existing human albumin, for example, according to the present invention, the prevention of the formation of particles with a diameter of 30-300 nm during storage is achieved , transportation or cultivation at ambient temperature as a result of aggregation or fibrillation of human albumin.

[0084] It should be understood that the term "particle" means a substance with a diameter within a certain range, and formed during storage, transportation and cultivation at ambient temperature of a medical product containing human albumin, as a result of aggregation or fibrillation of human albumin, where the preferred particle diameter according to the present invention is 30-300 nm.

[0085] The human albumin-containing medical product of the present invention can be used to suppress the formation of aggregates, agglomerates, and fibrillation by freezing and thawing in cold drying, freeze drying, and low temperature spray drying, and to accelerate recovery.

[0086] The human albumin-containing medical product of the present invention includes, but is not limited to, human serum-derived albumin obtained by the Kohn method and grinding, and recombinant human albumin purified by various steps.

[0087] The pH range of the medical product containing human albumin according to the present invention is 6-8, preferably 6.4-7.4. The pH values are given in the specifications according to each national pharmacopoeia.

[0088] The human albumin-containing medical product of the present invention may further contain diluents, buffers, solubilizers, excipients, pH adjusters, sulfur-containing reducing agents, antioxidants, and the like. Sulfur reducing agents include N-acetyl-DL-tryptophan (N-acetyltryptophan), N-acetyl-L-tryptophan, N-acetylmethionine, N-acetyl-L-methionine, etc. Commonly used buffers and diluents include sodium chloride solution, sodium phosphate buffer solution, sodium acetate solution and sodium citrate solution, preferably within the range specified in the specifications according to each national pharmacopoeia.

[0089] The human albumin-containing medical product of the present invention may further contain medium chain fatty acids such as sodium caprylate, sodium heptanoate, sodium decanoate, and preferably sodium caprylate according to the pharmacopeia specification.

[0090] The use of the human albumin-containing medical product of the present invention includes, but is not limited to, products such as injectable clinical preparations, culture media, drug excipients, medical devices, vaccine excipients, cosmetic products, and analytical diagnostic reagents.

[0091] Embodiments of the present invention are illustrated below with specific examples.

[0092] Embodiment 1

[0093] Commercial human serum albumin (sample 1, Shanghai RAAS blood products Co., Ltd., serial number: 201708A010) was used in the following dosage form: human albumin with excipients sodium caprylate and acetyltryptophan. This dosage form complies with pharmacopoeial requirements, i.e. 0.079 mmol of sodium caprylate was added per 1 g of albumin and 0.076 mmol of acetyltryptophan was added per 1 g of albumin, the pH was 6.8. The content of residual long chain fatty acids after purification of albumin originating from human serum was examined by gas chromatography. The following is a detailed description of the method for analyzing human albumin using gas chromatography (GC):

[0094] Gas Chromatography: Model: 7890A; manufacturer: Agilent Technologies Co., Ltd.

[0095] GC column: Acid-deactivated polyethylene glycol capillary column, TG-WAXMS A from Thermo Fisher Scientific Co., Ltd.; specification: 30 m × 0.32 mm × 0.25 µm;

[0096] Materials and reagents: heptadecanoic acid (C 17): category: US reference material; serial number: N-17A-JY16X; concentration: >100 mg; content: ≥99.0%; palmitic acid: category: national certified reference material; serial number: 190032-201603; concentration: 200 mg/ampoule; stearic acid: category: national certified reference material; serial number: 190032-201603; concentration: 200 mg/ampoule; oleic acid: category: national certified reference material; serial number: 111621-2015-06; concentration: 100 mg/ampoule; content: ≥99.0%; linoleic acid: category: US reference material; serial number: U-59A-J6-Y; concentration: >100 mg/ampoule; content: ≥99.0%; linolenic acid: category: US reference material; serial number: U-62A-S20-B; concentration: >100 mg/ampoule; content: ≥99.0%; chloroform: category: AR; serial number: 20170410; concentration: 500 ml/bottle; content: ≥99.0%; manufacturer: Sinopharm.

[0097] Experimental method: According to the Pharmacopeia of the People's Republic of China 2015 edition: an internal standard substance, heptadecanoic acid (C17), was added and the absolute peak area of palmitic acid, stearic acid, oleic acid, linoleic acid, linolenic acid was calculated acid and heptadecanoic acid (C17), built a linear regression with the ratio of the peak area of the reference solution of each fatty acid to the internal standard, depending on the amount of the specified reference solution of each fatty acid, obtained a linear regression equation and calculated the absolute peak area for each fatty acid in the test solution thus obtaining a calculated content of each fatty acid in the test sample.

[0098] Chromatography conditions: temperature: 260°C; column flow: 2.0 ml/min; purge flow: 20 ml/min; split flow: 2 ml/min; temperature program: initial hold for 5 min at 80°C, increase in temperature to 230°C at a rate of 10°C/min and hold for 25 min; in general 45 min; detector: model - FID; temperature: 280°C; hydrogen: 40 ml4 sh; air: 450 ml4 sh; make-up gas flow rate: 45 ml4 sch.

[0099] Accurate weighing of heptadecanoic acid (C 17) was carried out to obtain an internal standard solution; fatty acid standards were accurately weighed and these standards were mixed with the reference solution. Received a standard curve to calculate the content of fatty acids in human albumin.

[0100] The results of the study of the content of fatty acids in the preparation of human albumin for injection (Shanghai RAAS, 201703A010):

[0101] C16:0 content (palmitic acid) (mol/mol protein): 0.241

[0102] C18:0 (stearic acid) content (mol/mol protein): 0.147

[0103] C18:1 (oleic acid) content (mol/mol protein): 0.077

[0104] Content of C18:2 (linoleic acid) (mol/mol protein): 0.085

[0105] Content of C18:3 (linolenic acid) (mol/mol protein): not detected

[0106] Content of C20:4 (arachidonic acid) (mol/mol protein): not detected

[0107] The total content of the above acids: content of long chain fatty acids (mol/mol human albumin): 0.55:1

[0108] Dynamic light scattering was used to investigate:

[0109] Dynamic light scattering (DLS) analysis instrument model: Litesizer 500; manufacturer: Anton Paar (Shanghai) Trading Co., Ltd;

[0110] cuvettes: quartz cuvettes;

[0111] Sample: Bulk product sample solution was tested.

[0112] Method: measurement angle: automatic; temperature range: 25°C-60°C; step: 5°C.

[0113] Particles of 30-300 nm due to the instability of commercial human serum albumin must be removed using gel filtration; The following are the chromatography conditions: Carrier G-25 (General Electric Company), Chromatography apparatus: EV 50D chromatography system (Lisure Science (Suzhou) Co., Ltd.), Buffer system: Phosphoric acid solution, 0.1 mol/l, neutral pH, and purified water. The sample was concentrated using an ultrafiltration membrane (30K) after chromatography and was obtained as a product for research in accordance with the original dosage form of the product.

[0114] FIG. 1 is a chromatogram of the results of chromatography using DLS (DRS-chromatogram) of commercial human serum albumin (sample 1).

[0115] The 30 nm-300 nm particles in sample 1 were removed as described above and 75 μg of poloxamer 188 (BASF, serial number: WPAK527B) per 1 g of human albumin was added to obtain sample 2. The DLS chromatogram of sample 2 is shown in FIG. 2.

[0116] As shown by the results of the DLS in FIG. 1 and FIG. 2, microparticles between 30 nm and 300 nm were present in commercial poloxamer-free human serum albumin (Sample 1) at both 25°C and 60°C, whereas after particle removal, these microparticles were absent at 25°C. and 60°C in human serum albumin with a small amount of additional poloxamer (sample 2).

[0117] As a result of research, the inventors also found in accordance with the present application that, as a variation of this particular embodiment of the invention, poloxamer 188 can also be replaced by one of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407 and, alternatively, multiple poloxamers of poloxamer 188, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, or poloxamer can be used simultaneously. Studies have shown that the same effects can be achieved using these poloxamers, i.e. after removal of particles, microparticles with a diameter between 30 nm and 300 nm were absent both at 25°C and at 60°C in human serum albumin with a small amount of additional poloxamer.

[0118] As a result of the study, the inventors in accordance with the present application also found that the desired effect can be achieved by adding only a small amount of poloxamers. In general, when using an additional 10 μg or more poloxamers/gram human albumin, microparticles with a diameter between 30 nm and 300 nm were essentially not detected or were present only in a small amount; and with additional over 500 µg poloxamers/gram human albumin, a significant increase in microparticles between 30 nm and 300 nm in diameter was observed.

[0119] Embodiment 2

[0120] Commercial human serum albumin (sample 3, Shantou Weilun Bio-pharmaceutical Co., Ltd., serial number: 20170104) was used, in which the ingredients of its dosage form were human albumin with excipients sodium caprylate and sodium chloride. The dosage form complied with pharmacopoeial requirements, i.e. 0.159 mmol sodium caprylate was added per gram of albumin without additional N-acetyltryptophan. The pH value was 6.7 and the total sodium content did not exceed 160 mmol/l. The method according to embodiment 1 was adapted to determine the content of residual long chain fatty acids in sample 3. The content of long chain fatty acids (mol/mol human albumin) was 0.34:1.

[0121] FIG. 3 is a DLS chromatogram of sample 3.

[0122] The 30-300 nm particles in sample 3 were removed according to the method of embodiment 1 and 100 μg of poloxamer 188 (BASF, serial number WPAK527B) per 1 g of human albumin was added to obtain sample 4. DLS chromatogram of sample 4 shown in FIG. 4.

[0123] As shown by the DLS results in FIG. 3 and FIG. 4, microparticles between 30 nm and 300 nm were present in commercial human serum albumin without poloxamer at both 25°C and 60°C. After particle removal, microparticles were absent at 25°C and 60°C in human serum albumin with a small amount of additional poloxamer.

[0124] Embodiments 1 and 2 showed that about 200 nm diameter particles are present in both Pharmacopeia standard dosage forms of human serum albumin without the addition of poloxamer; in the case of a small amount of additional poloxamer, both effective suppression and prevention of the formation of microparticles can be observed.

[0125] As a result of research, the present inventors have also found that, as a variation of this particular embodiment, poloxamer 188 can also be replaced by one of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407, and alternatively, you can use several poloxamers from poloxamer 188, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 or poloxamer 407 simultaneously. after removal of particles, microparticles with a diameter between 30 nm and 300 nm were absent both at 25°C and at 60°C in human serum albumin with a small amount of additional poloxamer.

[0126] As a result of research, the inventors also found in accordance with the present application that the desired effect can be achieved by adding only a small amount of poloxamers. In general, when using an additional 10 μg or more of poloxamers/gram of human albumin, microparticles with a diameter between 30 nm and 300 nm were essentially not detected or were present only in a small amount; and when more than 500 μg additional poloxamers/gram human albumin were used, a significant increase in the number of microparticles with a diameter between 30 nm and 300 nm was observed.

[0127] Embodiment 3

[0128] In the present application, high purity bulk recombinant human albumin obtained after improved purification according to the method described in the Chinese patent application (application number 201010124935.2) was used. The investigated residual content of long chain fatty acids in purified recombinant human albumin, expressed as the ratio of the amount of indicated long chain fatty acids (mol/mol human albumin), was 0.059:1, which is about 10 times lower compared to the content of long chain fatty acids in albumin occurring from human serum. Sample 5 was prepared only according to the requirements for the method according to the Chinese Pharmacopoeia 2015 edition without the addition of any long chain fatty acids or poloxamers, i.e. 0.071 mmol of sodium caprylate (Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd, serial number 20170901) per 1 g of albumin and 0.085 mmol of acetyltryptophan (Adamas Reagent Co., Ltd, serial number P230264) per 1 g of albumin were added at a pH of 6 ,8, while the total sodium content was regulated so that it did not exceed 160 mmol/l. [0129] FIG. 5 is a DLS chromatogram of recombinant human albumin without the addition of poloxamer or long chain fatty acids (sample 5).

[0130] Embodiment 4

[0131] Preparation of Sample 6: High purity bulk recombinant human albumin prepared by the method of Embodiment 3 above was taken; adding sodium oleate (BBI Life Sciences, serial number: E802 BA0026) to the specified bulk product to achieve a molar ratio of 1.0:1.0 between long chain fatty acids and recombinant human albumin (counted residual long chain fatty acids in the bulk product); 0.081 mmol of sodium caprylate (Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd, serial number 20170901) per 1 g of albumin and 0.067 mmol of acetyltryptophan (Adamas Reagent Co., Ltd, serial number P230264) per 1 g of albumin were added at a pH of 6 ,7, while the total sodium content was regulated so that it did not exceed 160 mmol/l.

[0132] FIG. 6 is a DLS chromatogram of a medical product containing recombinant human albumin supplemented with long chain fatty acids only (sample 6).

[0133] Embodiment 5

[0134] Preparation of Sample 7: High purity bulk recombinant human albumin obtained by the method of embodiment 3 above was taken, 100 μg of poloxamer 188 (BASF, serial number WPAK527B) was added per 1 g of human albumin, 0.079 mmol of caprylate was added sodium (Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd, serial number 20170901) per 1 g of albumin and 0.066 mmol of acetyltryptophan (Adamas Reagent Co., Ltd, serial number P230264) per 1 g of albumin at a pH of 6.6, at In this case, the total sodium content was adjusted so that it did not exceed 160 mmol/l.

[0135] FIG. 7 is a DLS chromatogram of recombinant human albumin supplemented with poloxamer only (sample 7).

[0136] Embodiment 6

[0137] Preparation of Sample 8: The high purity bulk recombinant human albumin prepared by the method of Embodiment 3 above was taken, 100 µg of Poloxamer 188 (BASF, serial number WPAK527B) per 1 gram of human albumin was added, sodium oleate ( BBI Life Sciences, serial number: E802 BA0026) to achieve a molar ratio of 1.0:1.0 between long chain fatty acids and recombinant human albumin (counted residual long chain fatty acids in the bulk product), 0.083 mmol sodium caprylate (Chengdu Huayi Pharmaceutical Excipients) was added Manufacturing Co., Ltd, serial number 20170901) per 1 g of albumin and 0.084 mmol of acetyltryptophan (Adamas Reagent Co., Ltd, serial number P230264) per 1 g of albumin at a pH value of 6.8, while the total sodium content was adjusted as follows so that it does not exceed 160 mmol / l.

[0138] FIG. 8 is a DLS chromatogram of recombinant human albumin supplemented with poloxamer and long chain fatty acids (sample 8).

[0139] Embodiment 7

[0140] Preparation of Sample 9: High purity bulk recombinant human albumin obtained by the method of embodiment 3 above was taken, 100 μg of poloxamer 188 (BASF, WP serial number AK527 B) was added per 1 g of human albumin, soybeans were added sodium (Qingdao Raynol Chemical Co., Ltd., serial number 20190121) to achieve a molar ratio of 0.5:1.0 between long-chain fatty acids and recombinant human albumin (the residual long-chain fatty acids were considered in the bulk product), 0.082 mmol of sodium caprylate was added (Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd, serial number 20170901) per 1 g of albumin and 0.080 mmol of acetyltryptophan (Adamas Reagent Co., Ltd, serial number P230264) per 1 g of albumin at a pH of 6.9, while the total the sodium content was regulated so that it did not exceed 160 mmol/l.

[0141] FIG. 9 is a DLS chromatogram of recombinant human albumin supplemented with poloxamer and long chain fatty acid (sodium soyate) (sample 9).

[0142] Embodiment 8

[0143] Preparation of Sample 10: High purity bulk recombinant human albumin obtained by the method of embodiment 3 above was taken, 50 μg of poloxamer 188 (BASF, serial number WPAK527B) was added per 1 g of human albumin, sodium stearate was thoroughly mixed (Damas-beta, serial number: P1042477) and sodium oleate (BBI Life Sciences, serial number: E802 BA0026) in a 1:2 molar ratio and added to the bulk product to achieve a 0.6:1.0 molar ratio between the mixed long chain fatty acids and recombinant human albumin (considered residual long chain fatty acids in the bulk product), 0.086 mmol sodium caprylate (Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd, serial number 20170901) per 1 g of albumin and 0.076 mmol acetyltryptophan (Adamas Reagent Co., Ltd, serial number P230264) per 1 g of albumin at a pH of 7.0, while the total sodium content was adjusted so that it did not exceed 160 mmol/l.

[0144] FIG. 10 is a DLS chromatogram of recombinant human albumin with additional poloxamer and long chain fatty acids (mixture of sodium stearate and sodium oleate) (sample 10).

[0145] Embodiment 9

[0146] Preparation of Sample 11: High purity bulk recombinant human albumin obtained by the method of embodiment 3 above was taken, 50 μg of poloxamer 188 (BASF, serial number WPAK527B) was added per 1 g of human albumin, sodium stearate was thoroughly mixed (Damas-beta, serial number: P1042477) and sodium oleate (BBI Life Sciences, serial number: E802 BA0026) in a 1:2 molar ratio and added to the bulk product to achieve a 0.6:1.0 molar ratio of the long chain fatty acid mixture and recombinant human albumin (the residual long chain fatty acids in the bulk product were considered), 0.153 mmol sodium caprylate (Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd, serial number 20170901) was added per 1 g of albumin at a pH of 7.0, with a total the sodium content was regulated so that it did not exceed 160 mmol/l.

[0147] FIG. 11 is a DLS chromatogram of recombinant human albumin supplemented with poloxamer and long chain fatty acid (sodium stearate/sodium oleate mixture) (sample 11). [0148] Embodiment 10

[0149] Incubation and study of thermal stability of samples obtained according to embodiments of the invention 1-9 (Samples 1-11). These methods were carried out in accordance with the Chinese Pharmacopoeia 2015 edition, volume III, pages 244-245. The results are shown in Table 1.

[0150] Table 1. Results of monitoring the stability of samples 1-11

[0151] The above embodiments have demonstrated the effect of oleic acid, sodium soybean, a mixture of sodium stearate and sodium oleate, and a mixture of sodium stearate and sodium oleate in combination with poloxamer in providing stability to recombinant human albumin products. As a result of research, the inventors have also found in accordance with the present application that, as a variation of the described embodiments of the invention, long chain fatty acids can also be one or more acids selected from myristic acid (C14:0), palmitic acid (C16 :0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3) and arachidonic acid (C20:4). Studies have shown that the same effect can be achieved using these long chain fatty acids, ie. their use in combination with poloxamers can provide stability to recombinant human albumin products. Negative responses were shown in the above stability study.

[0152] As a result of research, the inventors also found in accordance with the present application that, as a variation of the above embodiments of the invention, poloxamer 188 can also be replaced by one of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 and poloxamer 407 , and, alternatively, several of poloxamer 188, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, or poloxamer 407 can be used simultaneously. Studies have shown that the same effect is achieved using these poloxamers; the stability of human serum albumin can be met with the addition of some long chain fatty acids and a small amount of poloxamer.

[0153] As a result of research, the inventors in accordance with the present application also found that the desired effect can be achieved by adding only a small amount of poloxamer. In general, stability monitoring results essentially show a negative response with the addition of 10 µg or more poloxamer/g human albumin, but with the addition of more than 500 µg poloxamer/g human albumin, stability studies begin to show positive responses.

[0154] As a result of the study, the inventors also found in accordance with the present application that the stability of the medical product of human albumin is best when the molar ratio between long chain fatty acids and human albumin is 0.2-3.0, and in particular, 0 .3-2.0. When the molar ratio between long chain fatty acids and human albumin is less than 0.3 or more than 2.0, a small number of positive responses begin to be detected in stability studies, whereas when the molar ratio between long chain fatty acids and human albumin is less than 0, 2 or more than 3.0, a large number of

positive responses.

[0155] Embodiment 11

[0156] Samples 5-11 obtained using the method according to embodiments 3-9 were analyzed by differential scanning calorimetry (DSC) and chromatograms were obtained from the results of chromatography using DSC (DSC chromatogram). DSC instrument model: Nano DSC 602000; manufacturer: TA; sample diluent: 0.01 mol/l pH neutral phosphoric acid. Test conditions: Bulk product sample was diluted with diluent to a concentration of 1-5 mg/mL; temperature range: 45°C - 90°C; step: 1°C/min. The results are shown in Table 2.

[0157] Table 2. DSC results of samples 5-11

[0158] The above results according to the present invention showed a synergistic stabilizing effect of poloxamers and long chain fatty acids. 30-300 nm and improve thermal stability by adding only a small amount of additional poloxamers without the need for additional addition of long chain fatty acids.

[0159] For high purity recombinant human albumin, which is essentially completely defatted, additional poloxamers are required to suppress the appearance of particles with a diameter of 30-300 nm. Comparison of the results of the study shows that in the case where only poloxamers, standard pharmacopoeial sodium caprylate (medium chain fatty acids) and acetyltryptophan are added, the requirements for thermal stability cannot be met due to the lack of mutual support of various ligands of adjacent albumin domains provided by long-chain fatty acids, despite the fact that medium chain fatty acids can effectively increase T _onset , T _m1 and T _m2 .

[0160] Thus, the regulation of long chain fatty acids can effectively increase T _onset , T _m1 and T _m2 , and since the interrelated stability of protein domains is increased, a small amount of additional poloxamers is required to suppress hydrophobic interactions. The human albumin-containing medical product of the present invention meets the requirements for stability when incubated at 57°C for 50 hours, at 30°C for 14 days, and at 20°C for 28 days with excellent stability, meeting the stability requirements for long-term storage and transportation at 2-8°C and room temperature.

[0161] The embodiments of the invention described above are provided solely for the purpose of illustrating the principles of the invention and their effectiveness and do not limit the present invention. A person skilled in the art can make modifications or changes to the above embodiments, provided that they do not contradict the essence and scope of the present invention. Thus, all equivalent modifications or changes made by one of ordinary skill in the art within the spirit and technical concept described in accordance with the present invention are included in the claims of the present invention.

Claims (20)

1. A preparation containing human albumin, wherein said preparation contains poloxamer and fatty acids, while the molar ratio of fatty acids and human albumin in the specified preparation is 0.5-2.0:1.0, the content of poloxamer in the indicated preparation is 10-100 μg of poloxamer / 1 g of human albumin, where said poloxamer is one or more poloxamers selected from poloxamer 188 and poloxamer 407, where said fatty acid is one or more of myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0) and oleic acid (C18:1), where human albumin is human serum albumin or human recombinant albumin. 2. A preparation according to claim 1 containing human albumin, wherein said fatty acid is soy fatty acid or fully hydrogenated soy fatty acid. 3. The drug according to claim 1, containing human albumin, where the specified drug additionally contains ingredients in quantities in accordance with national pharmacopoeias, including sodium caprylate and/or N-acetyltryptophan. 4. A method for obtaining a preparation containing human albumin, including: providing human albumin, adding 10-100 µg of poloxamer per 1 g of human albumin, and adding fatty acids to achieve a molar ratio of fatty acids to human albumin equal to 0.5-2.0:1.0, where said poloxamer is one or more poloxamers selected from poloxamer 188 and poloxamer 407, where said fatty acid is one or more of myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0) and oleic acid (C18:1), where human albumin is human serum albumin or human recombinant albumin. 5. The method according to p. 4, where the specified method further includes: adding sodium caprylate and/or N-acetyltryptophan in accordance with national pharmacopoeias. 6. The preparation according to claim 1 containing human albumin, while in the specified preparation there is no turbidity or visible particles during incubation at 57°C for 50 hours, incubation at 30°C for 14 days and incubation at 20°C for within 28 days. 7. The method of claim 4 wherein said fatty acids are added in the form of salts. 8. The method of claim 4 wherein said fatty acids are added in the form of sodium salts. 9. The method according to p. 4, where the specified preparation does not contain particles with a diameter of more than 30-200 nm, formed as a result of aggregation and / or fibrillation of human albumin.