WO2021051807A1 - 含人白蛋白的制剂及其制备方法 - Google Patents

含人白蛋白的制剂及其制备方法 Download PDF

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WO2021051807A1
WO2021051807A1 PCT/CN2020/086057 CN2020086057W WO2021051807A1 WO 2021051807 A1 WO2021051807 A1 WO 2021051807A1 CN 2020086057 W CN2020086057 W CN 2020086057W WO 2021051807 A1 WO2021051807 A1 WO 2021051807A1
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preparation
human albumin
poloxamer
acid
chain fatty
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PCT/CN2020/086057
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English (en)
French (fr)
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项炜
岳志蕾
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通化安睿特生物制药股份有限公司
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Priority to US17/642,239 priority Critical patent/US20220313790A1/en
Priority to JP2022510834A priority patent/JP7414326B2/ja
Priority to KR1020227007537A priority patent/KR20220061964A/ko
Priority to CA3154018A priority patent/CA3154018A1/en
Priority to EP20866272.6A priority patent/EP4032541A4/en
Priority to BR112022004994A priority patent/BR112022004994A2/pt
Publication of WO2021051807A1 publication Critical patent/WO2021051807A1/zh

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A61K38/385—Serum albumin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P17/00—Drugs for dermatological disorders
    • A61P17/02—Drugs for dermatological disorders for treating wounds, ulcers, burns, scars, keloids, or the like
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/17—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • A61K38/38—Albumins
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/10—Alcohols; Phenols; Salts thereof, e.g. glycerol; Polyethylene glycols [PEG]; Poloxamers; PEG/POE alkyl ethers
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K47/00—Medicinal preparations characterised by the non-active ingredients used, e.g. carriers or inert additives; Targeting or modifying agents chemically bound to the active ingredient
    • A61K47/06—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite
    • A61K47/08—Organic compounds, e.g. natural or synthetic hydrocarbons, polyolefins, mineral oil, petrolatum or ozokerite containing oxygen, e.g. ethers, acetals, ketones, quinones, aldehydes, peroxides
    • A61K47/12—Carboxylic acids; Salts or anhydrides thereof
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/08—Solutions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K9/00—Medicinal preparations characterised by special physical form
    • A61K9/14—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles
    • A61K9/19—Particulate form, e.g. powders, Processes for size reducing of pure drugs or the resulting products, Pure drug nanoparticles lyophilised, i.e. freeze-dried, solutions or dispersions
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P1/00—Drugs for disorders of the alimentary tract or the digestive system
    • A61P1/16—Drugs for disorders of the alimentary tract or the digestive system for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P13/00—Drugs for disorders of the urinary system
    • A61P13/12—Drugs for disorders of the urinary system of the kidneys
    • 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
    • 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
    • A61P7/04—Antihaemorrhagics; Procoagulants; Haemostatic agents; Antifibrinolytic agents
    • 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
    • A61P7/08—Plasma substitutes; Perfusion solutions; Dialytics or haemodialytics; Drugs for electrolytic or acid-base disorders, e.g. hypovolemic shock
    • 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
    • A61P7/10—Antioedematous agents; Diuretics
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/02—Non-specific cardiovascular stimulants, e.g. drugs for syncope, antihypotensives
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • the invention relates to the field of biomedicine, in particular to a preparation containing human albumin and a preparation method thereof.
  • human albumin includes human serum albumin (Human Serum Album) and recombinant human albumin (Recombinant Human Album).
  • Recombinant human albumin is also called Recombinant Human Serum Albumin, or Recombinant Human Albumin Mutant.
  • human albumin The main pharmacological effects of human albumin include regulating the dynamic balance of water between tissues and blood vessels, maintaining a normal and constant plasma volume, and at the same time having a high affinity for certain ions and compounds, reversibly binding with these substances, and exerting a transport function.
  • Human albumin also provides a large amount of amino acid reserves for the body. Due to the above-mentioned effects of human albumin, it can be used in various clinical disciplines and exert a variety of therapeutic effects. Human albumin is clinically mainly used to regulate plasma colloidal osmotic pressure, expand blood volume, treat trauma, hemorrhagic shock, severe burns and hypoproteinemia. It is widely used in common diseases such as stroke, liver cirrhosis, liver ascites, and kidney disease. application. In addition to directly in the field of clinical treatment, albumin has also been widely used in culture media, pharmaceutical excipients, diagnostic reagents, new tumor long-acting products, cosmetics, laboratory biological reagents and other aspects used in vaccine production.
  • human albumin is a single-chain non-glycosylated protein with a heart-shaped structure, with 585 amino acids, 17 pairs of disulfide bonds, a free sulfhydryl group, and a molecular weight of 66438 Daltons.
  • the half-life of human albumin in the human body is 19-21 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. It can be seen from its crystal structure that the disulfide bridge imparts rigidity to the helical spherical structure, but provides enough 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 separate, purify, extract and purify from human serum, collectively referred to as human serum albumin.
  • human serum albumin The discovery and use of human serum albumin has gone through decades, and it has developed into a complete set of production and preparation processes (Cohn method-ultrafiltration-/or purification-pasteurization-preparation (sodium caprylate and/or N- Acetyltryptophan)), its preparation prescription has become the general standard of the pharmacopoeias of all countries in the world.
  • Albumin derived from human serum has good stability. The pharmacopoeia requires that the finished product be kept in a water bath at 57°C ⁇ 0.5°C for 50 hours.
  • the instructions of some manufacturers of human serum albumin specify the requirements for the use of precision infusion devices. Even so, the particle size of the above-mentioned partially unstable albumin is between 30 nm and 100 nm, and the general sterilization filter membrane (0.1 ⁇ m to 0.22 ⁇ m) cannot be effectively removed. Aggregates of this diameter are between multimers and visible foreign bodies, and are the main cause of adverse reactions such as immunogenicity.
  • human albumin Another source of human albumin is through genetic recombination to cultivate a host that can specifically express human albumin, such as fungal eukaryotic single-celled Pichia pastoris or Saccharomyces cerevisiae, or rice.
  • human albumin expressed by transgene.
  • the highly purified finished product of recombinant human albumin adopts the same preparation requirements as those stipulated in the pharmacopoeia, and its stability is far less than that of human albumin derived from human serum.
  • polysorbate 80 The negative effect of polysorbate is that the residual alkyl oxides in nonionic surfactants may increase the possibility of catalyzing the oxidation of proteins.
  • CNTF recombinant human ciliary neurotrophic factor
  • rhG-CSF recombinant human granulocyte colony stimulating factor
  • Human albumin contains methionine and a free sulfhydryl group, which can resist the peroxidation of free radicals in the body and prevent cells and organs from being damaged. Therefore, the quality and storage conditions of polysorbates need to be well controlled, and the correlation between their use in protein needs to be kept to a minimum (AGGREGATION OF THERAPEUTIC PROTEINS, P341, Edited by Wei Wang, Christopher J. Roberts) , WILEY2010), otherwise human albumin will be partially oxidized and lose its redox physiological activity. At the same time, oxidized human albumin will further promote albumin unfolding and accelerate the agglomeration and fibrosis of human albumin.
  • Human albumin has a very important physiological role in the circulation of the body, which depends on the ligand binding properties to carry drugs, fatty acids, vitamins, cytochromes and metal ions.
  • fatty acid has a decisive effect on 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 T m (melting temperature) value, thereby increasing the thermal stability.
  • DSC differential scanning calorimetry
  • human serum-derived albumin In addition to fatty acids, human serum-derived albumin also has ligands such as bilirubin, hormones, fat-soluble drugs, blood red and other ligands that are tightly bound to albumin, increasing the configuration stability of albumin.
  • ligands such as bilirubin, hormones, fat-soluble drugs, blood red and other ligands that are tightly bound to albumin, increasing the configuration stability of albumin.
  • albumin derived from human serum is defatted, its stability drops sharply, which is completely consistent with highly purified recombinant human albumin.
  • surfactants such as Tween should be added as little as possible to avoid their toxic effects. More importantly, they should reduce the interference of surfactants such as Tween on the physiological effects of human albumin ligands, or find more Safe and lower dosage of surfactants.
  • Poloxamer is the general name of polyoxyethylene-polyoxypropylene copolymer. The trade name produced by BASF in Germany is Pluronic, which is a non-ionic surfactant. Poloxamer is currently the only synthetic emulsifier used in intravenous emulsions. Poloxamer 188 has the best emulsification performance and safety. It is an O/W emulsifier with a usual concentration of 0.3% (Wang Meng et al. , Pharmacy and Clinical Research, 2007 Vol. 15, Issue 1, Pages 10-13).
  • Poloxamer 188 can increase the solubility of poorly soluble drugs by forming micelles, and its critical micelle concentration (CMC) is about 0.6%. Poloxamer 188 can inhibit thrombus formation, affect hemorheological activities, seal cell membranes, activate phagocytes (stimulated phagocytic function and super-negative ion production) and neutrophil degranulation, and prevent adult skeletal muscle necrosis . Poloxamer 188 can also treat brain injury: Studies have shown that Poloxamer 188 can reduce inflammation and tissue damage caused by experimental brain injury in rats. There is also evidence in other experimental models that it can fight tissue damage. The protective mechanism may involve the effect of surfactants on oxidative stress and inflammation.
  • Poloxamer 188 can also improve cell survival. Studies have shown that a single dose of intravenous poloxamer 188 can effectively restore damaged tissues by establishing a complete circulation. Poloxamer 188 can stabilize cell membrane defects caused by many reasons. Studies in rats have shown that a single dose of poloxamer 188 can combat early neuronal damage after bleeding, but has no effect on neuronal damage caused by long-term bleeding. However, daily administration of poloxamer 188 may produce long-term, effective Neuron protection.
  • the Chinese patent application with application number CN200480003079.8 discloses that poloxamer is used as a surfactant to stabilize hematopoietic factors, parathyroid hormone or antibodies.
  • the protein in this patent does not have the role of fatty acid ligands, so it adds a larger dose
  • hematopoietic factors, parathyroid hormone or antibodies are small-volume injections (ug ⁇ mg/bottle), even if a larger concentration of poloxamer is added, the total amount of poloxamer is not large.
  • human albumin is a large-volume injection, and the injection dose is 5 to 12.5 g/bottle. Even if a small concentration of Tween or poloxamer is added, the total amount of Tween or poloxamer is relatively large.
  • the technical problem to be solved by the present invention is to increase the stability of the human albumin preparation.
  • the present invention provides a method for preparing a preparation containing human albumin.
  • the method includes: controlling the content of long-chain fatty acids in the preparation and adding poloxamer to improve the The stability of human albumin in the formulation is described.
  • the method further comprises: improving the stability of the human albumin in the preparation by reducing the agglomeration and fibrillation of the human albumin.
  • the method further comprises: improving the stability of the human albumin in the preparation so that no particles with a particle size of 30 nm or more formed by agglomeration and/or fibrillation of human albumin appear after the preparation of the preparation is completed .
  • the method further includes: improving the stability of the human albumin in the preparation so that no particles with a particle size above 100 nm formed by agglomeration and/or fibrillation of human albumin appear after the preparation of the preparation is completed .
  • the method further comprises: improving the stability of the human albumin in the preparation so that no particles with a particle size of about 200 nm formed by agglomeration and/or fibrillation of human albumin appear after the preparation of the preparation is completed .
  • the method further comprises: improving the stability of the human albumin in the preparation so that the stability meets the requirements of the local pharmacopoeia.
  • the method further includes: increasing the stability of the human albumin in the formulation so that the stability reaches the following conditions: no turbidity and turbidity appear during incubation at 57°C for 50h, 30°C for 14 days, and 20°C for 28 days. Foreign objects can be seen.
  • the human albumin is human serum albumin
  • the method includes: increasing the stability of the human albumin in the preparation by adding poloxamer.
  • the human albumin is recombinant human albumin, and the method includes: increasing the stability of the human albumin in the preparation by adding long-chain fatty acids and poloxamers.
  • the long-chain fatty acid is added in the form of a salt.
  • the long-chain fatty acid is added as a sodium salt.
  • the method further includes: after the preparation of the preparation is completed, the molar ratio of the long-chain fatty acid to the human albumin is 0.2-3.0.
  • the method further includes: after the preparation of the preparation is completed, the molar ratio of the long-chain fatty acid to the human albumin is 0.3-2.0.
  • the method further includes: after the preparation of the preparation is completed, the content of the poloxamer is 10-500 micrograms/g human albumin.
  • the poloxamer is selected from one or more of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407.
  • the poloxamer is poloxamer 188.
  • the long-chain fatty acid is selected from the group consisting of myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18: 2) One or more of linolenic acid (C18:3) and arachidonic acid (C20:4).
  • the long-chain fatty acid is soybean oleic acid or fully hydrogenated soybean oleic acid.
  • the method further includes: adding ingredients and contents required by the local pharmacopoeia.
  • the ingredients required by the local pharmacopoeia include sodium caprylate or/and N-acetyl tryptophan.
  • the invention also provides a human albumin-containing preparation prepared according to the method provided by the invention.
  • the present invention also provides a preparation containing human albumin, which does not contain particles with a particle size above 30 nm formed by agglomeration and/or fibrillation of human albumin.
  • the present invention also provides a preparation containing human albumin, which does not contain particles with a particle size above 100 nm formed by agglomeration and/or fibrillation of human albumin.
  • the present invention also provides a preparation containing human albumin, which does not contain particles with a particle size of about 200 nm formed by agglomeration and/or fibrillation of human albumin.
  • the present invention also provides a preparation containing human albumin, and the method further comprises: the stability of the human albumin in the preparation reaches the following conditions: incubate at 57°C for 50 hours, incubate at 30°C for 14 days, and incubate at 20°C No turbidity or visible foreign matter appeared in 28 days.
  • the present invention also provides a preparation containing human albumin, which contains poloxamer and long-chain fatty acids.
  • the molar ratio of long-chain fatty acid to human albumin in the preparation is 0.2-3.0.
  • the molar ratio of long-chain fatty acid to human albumin in the preparation is 0.3-2.0.
  • the method further comprises: the content of the poloxamer in the preparation is 10-500 micrograms/g human albumin.
  • the poloxamer is selected from one or more of poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338, and poloxamer 407.
  • the poloxamer is poloxamer 188.
  • the long-chain fatty acid is selected from the group consisting of myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18: 2) One or more of linolenic acid (C18:3) and arachidonic acid (C20:4).
  • the long-chain fatty acid is soybean oleic acid or fully hydrogenated soybean oleic acid.
  • the preparation also contains the ingredients and contents required by the local pharmacopoeia.
  • the ingredients required by the local pharmacopoeia include sodium caprylate or/and N-acetyl tryptophan.
  • the present invention is based on fatty acid ligands to increase the thermal denaturation stability of human albumin.
  • Poloxamer acts as a hydrophobic patch to shield hydrophobic interactions and interface hydrophobic interactions to reduce agglomeration and fibrillation, thereby improving low temperature, normal temperature, high temperature or Stability caused by agglomeration and fibrillation during shock transportation.
  • the diameter of such agglomerates/aggregates and fibrillated particles is between 30nm-300nm, mainly between 100-200nm, which cannot be detected by HPLC-SEC.
  • Membrane systems of 0.1 ⁇ m ⁇ 0.2 ⁇ m cannot effectively filter.
  • the unstable albumin solution system (even if the agglomerates/aggregates and fibrillated particles are removed) will continue to be produced during storage and transportation, and the diameter will increase until the micron and sub-millimeter diameters are visible to the naked eye. .
  • the inventors of the present application conducted extremely detailed studies to analyze albumin derived from human blood and found that every mole of albumin derived from human blood Contains 0.3 to 0.8 mol of long-chain fatty acids.
  • the human albumin circulating in the human body contains 0.5 to 2.5 mol of long-chain fatty acids per mole, which plays the physiological function of fatty acid mobilization and storage. Some long-chain fatty acids are lost in the process of producing and purifying albumin from human blood.
  • recombinant human albumin Regardless of the method of expression of recombinant human albumin, purification processes such as multi-step separation, chromatography, ultrafiltration and dialysis are required to completely remove host proteins, polysaccharides, endotoxins, and various pollutions, while also blending fatty acids
  • the body is removed cleanly and becomes defatted human albumin. Therefore, the stability of recombinant human albumin after the loss of fatty acids and other ligands in the human body is much lower than that of human blood-derived albumin. Therefore, a certain amount of surfactant is appropriately added, but its concentration is lower than the concentration of CMC.
  • the role of the surfactant is to play a hydrophobic patch of albumin that has lost its ligand to prevent albumin/air/container/vibration. Agglomeration after unfolding caused by various hydrophobic interface interactions such as bubbles.
  • the present invention adds a certain amount of poloxamer and adjusts the ratio of long-chain fatty acids on the basis of the preparation general rules of the Chinese Pharmacopoeia, the United States Pharmacopoeia, and the European Pharmacopoeia and human serum albumin, so that human albumin is regulated in the pharmacopoeias Meet the stability requirements under the conditions.
  • the advantage of the present invention is that it can directly add long-chain fatty acid ligands to the human body circulation in a reasonable proportion and a small amount of poloxamer, or use existing human albumin conventional prescriptions, and then add a small amount of poloxamer to make human white
  • the thermal stability of the protein and no aggregation or fibrillation occurs during storage and transportation.
  • the diameter of this kind of agglomerate/aggregate and fibrillation is between 50nm ⁇ 300nm, mainly between 100 ⁇ 200nm, which cannot be detected by HPLC-SEC.
  • Membrane system of 0.1 ⁇ m ⁇ 0.2 ⁇ m cannot filter effectively, which is more important. It is that the unstable albumin solution system (even if the agglomerates are removed) will continue to be produced during storage and transportation, and the diameter will increase until the micron and sub-millimeter diameters are visible to the naked eye.
  • Figure 1 is a dynamic light scatterer (DLS) diagram of sample 1 (human serum albumin containing sodium caprylate and acetyl tryptophan) in Example 1.
  • DLS dynamic light scatterer
  • Figure 2 is the DLS profile of sample 2 (human serum albumin containing sodium caprylate and acetyltryptophan, with poloxamer added) in Example 1.
  • Figure 3 is the DLS profile of sample 3 (commercially available human serum albumin containing only sodium caprylate) in Example 2.
  • Figure 4 is the DLS profile of sample 4 in Example 2 (commercially available human serum albumin containing only sodium caprylate and poloxamer added).
  • Figure 5 is the DLS profile of sample 5 (recombinant human albumin containing sodium caprylate and acetyltryptophan without adding poloxamer and long-chain fatty acids) in Example 3.
  • Figure 6 shows the DLS profile of sample 6 (recombinant human albumin containing sodium caprylate and acetyltryptophan, only long-chain fatty acid (oleic acid) added) in Example 4. .
  • Figure 7 is the DLS profile of sample 7 (recombinant human albumin containing sodium caprylate and acetyltryptophan, only poloxamer added) in Example 5.
  • Figure 8 is the DLS profile of sample 8 (recombinant human albumin containing sodium caprylate and acetyltryptophan, poloxamer and long-chain fatty acid (oleic acid)) in Example 6.
  • Figure 9 shows the DLS profile of sample 9 (recombinant human albumin containing sodium caprylate and acetyltryptophan, poloxamer and long-chain fatty acid (sodium soybean oleate)) in Example 7.
  • Figure 10 is the DLS profile of sample 10 (recombinant human albumin containing sodium caprylate and acetyltryptophan, added poloxamer and long-chain fatty acids (sodium stearate/sodium oleate mixed)) in Example 8.
  • Figure 11 is the DLS profile of sample 11 (contained sodium caprylate, poloxamer added and long-chain fatty acid (sodium stearate/sodium oleate mixed) recombinant human albumin) in Example 9.
  • human albumin refers to human serum albumin and/or recombinant human albumin.
  • Recombinant human albumin is also called recombinant human serum albumin, or is a recombinant human albumin mutant.
  • Recombinant human albumin includes at least microbial genetically recombined human albumin, eukaryotic recombinant human albumin, plant transgenic recombinant human albumin, animal transgenic recombinant human albumin, and the like.
  • preparation should be understood to include at least solution preparations, freeze-dried preparations or low-temperature spray-dried preparations, preferably solution preparations, which are usually aseptically filled in glass or plastic containers of a definite volume.
  • human albumin-containing preparations should be understood as preparations with different human albumin concentrations suitable for clinical injections, culture media, pharmaceutical excipients, medical devices, vaccine excipients, cosmetics, analytical and diagnostic reagents, etc.
  • concentration of human albumin is 1%-30%, preferably 10%-25%.
  • Poloxamer is a nonionic surfactant, a block copolymer of ethylene oxide and 1,2-propylene oxide. Its general formula is H(C2H4O) a (C3H6O) b (C2H4O) a OH. Poloxamer 188 is listed as an emulsifier for intravenous fat emulsions and a stabilizer for antibody injections in the 2015 edition of the Chinese Pharmacopoeia, US Pharmacopoeia, European Pharmacopoeia, and approved drugs. It has high biological safety.
  • long-chain fatty acid refers to fatty acids with more than 12 carbon atoms on the carbon chain, and should be understood as fatty acids from various sources (synthetic or extracted) acceptable to the human body, or fatty acids in various forms (for example, salts, Especially sodium salt), including but not limited to myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oleic acid (C18:1), linoleic acid (C18: 2) One or more of linolenic acid (C18:3) and arachidonic acid (C20:4).
  • control is the content of long-chain fatty acids in the preparations containing human albumin, which means that the amount of long-chain fatty acids in the preparations containing human albumin is maintained within a given range: when prepared by existing methods
  • the human albumin-containing preparations contain long-chain fatty acids, such as human blood-derived preparations containing human albumin. If the content of long-chain fatty acids is lower than a given range, “control” means that the preparation of human albumin-containing preparations The long-chain fatty acids are added during the preparation process so that the long-chain fatty acids in the final human albumin-containing preparation are within the given range.
  • control means that the preparation of human albumin-containing The preparation of albumin does not require any operation; when the preparation containing human albumin prepared by the existing method does not contain or contains a very small amount of long-chain fatty acids, "control” refers to the preparation of the preparation containing human albumin The long-chain fatty acid is added during the process so that the long-chain fatty acid in the final human albumin-containing preparation is within a given range.
  • long-chain fatty acid should be understood as a fixed phrase in which the "or” has no logical relationship with the context of this phrase.
  • content of “long-chain fatty acid” refers to the total amount of long-chain fatty acid, which is To make the content of long-chain fatty acids within a given range, long-chain fatty acids can be added, or a mixture of long-chain fatty acids can be added.
  • content of long-chain fatty acids should be understood as the content of long-chain fatty acids in the final human albumin-containing preparation, including the amount of long-chain fatty acids remaining in the preparation process due to the raw material itself containing long-chain fatty acids and extra The amount of long-chain fatty acids added (if added during the "control" process).
  • addition when the main body is poloxamer, should be understood as adding poloxamer during the preparation of the human albumin-containing formulation so that the poloxamer in the final human albumin-containing formulation Hm is within the given range.
  • control and “addition” should be understood as being implemented during the preparation of the human albumin-containing preparation, so that the content of long-chain fatty acids and the content of poloxamer in the final prepared human albumin-containing preparation are equal.
  • it can be implemented in the semi-finished product of the preparation, or through the ultrafiltration and dialysis process, or it can be added as a protein stabilizer before pasteurization, or in the last step of the purification chromatography process.
  • the purpose is to implement the final steps in order to achieve the final preparation containing human albumin to obtain the content range of poloxamer and long-chain fatty acid specified in the present invention.
  • the term "improvement” should be understood as the stability of the human albumin-containing preparation of the present invention is higher than that of the existing human albumin.
  • the present invention realizes the storage, transportation, and normal temperature cultivation process of the human albumin-containing preparation. There are no particles with a particle size of 30nm-300nm formed by human albumin agglomeration/aggregation or fibrillation.
  • particle should be understood as a substance with a certain particle size range formed by human albumin agglomeration/aggregation or fibrillation during storage, transportation, and normal temperature incubation of preparations containing human albumin.
  • the present invention The preferred particle size range is 30nm-300nm.
  • the human albumin-containing preparation of the present invention can be used to inhibit polymer, agglomerate and fibrosis formed by freeze-thaw of freeze-drying, freeze-drying and low-temperature spray drying, and accelerate reconstitution.
  • the human albumin-containing preparations of the present invention include, but are not limited to, albumin derived from human serum manufactured by the Cohn method and chromatographic purification, and recombinantly expressed human albumin purified through different steps.
  • the pH value of the human albumin-containing preparation of the present invention is in the range of 6-8, preferably the pH value of 6.4-7.4 as regulated by the national pharmacopoeia.
  • the human albumin-containing preparation of the present invention may also include diluents, buffers, solubilizers, excipients, pH adjusters, sulfur-containing reducing agents, antioxidants, and the like.
  • Sulfur-containing reducing agents include N-acetyl DL-tryptophan (N-acetyltryptophan), N-acetyl-L-tryptophan, N-acetylmethionine, N-acetyl-L-methionine and the like.
  • Commonly used buffers and diluents include sodium chloride solution, sodium phosphate buffer solution, sodium acetate solution, sodium citrate solution, preferably in the range of the pharmacopoeia specifications of various countries.
  • Medium-chain fatty acids such as sodium caprylate, sodium heptanoate, and sodium caprate, can also be added to the human albumin-containing preparation of the present invention, preferably sodium caprylate regulated by the Pharmacopoeia.
  • the human albumin-containing preparations of the present invention include, but are not limited to, applications in clinical injections, culture media, pharmaceutical excipients, medical devices, vaccine excipients, cosmetics, analytical diagnostic reagents, and so on.
  • human serum albumin (Sample 1, Shanghai RAAS Blood Products Co., Ltd., batch number: 201708A010)
  • the prescription is: human serum albumin
  • the excipients are sodium caprylate and acetyl tryptophan.
  • the prescription meets the requirements of the Pharmacopoeia, adding 0.079mmol of sodium caprylate per 1g of albumin and 0.076mmol of acetyltryptophan per 1g of albumin, and the pH is 6.8.
  • Gas chromatography was used to test the residual long-chain fat content of human albumin derived from serum after purification.
  • the specific method for analyzing human albumin by gas chromatography is as follows:
  • Chromatographic column acid-modified polyethylene glycol capillary column TG-WAXMS A Thermo Scientific Co., Ltd.; specification: 30m*0.32mm*0.25 ⁇ m;
  • C17 alkanoic acid grade: USA standard material; batch number: N-17A-JY16X; specification: >100mg; content: ⁇ 99.0%; palmitic acid: grade: national standard material; batch number: 190032-201603; specification: 200mg/piece; stearic acid: grade: national standard material; batch number: 190032-201603; specification: 200mg/piece; oleic acid: grade: national standard material; batch number: 111621-2015-06; specification: 100mg/piece; content : ⁇ 99.0%; linoleic acid: grade: USA standard material; batch number: U-59A-J6-Y; specification: >100mg/piece; content: ⁇ 99.0%; linoleic acid: grade: USA standard material; batch number: U-62A-S20-B; Specification: >100mg/piece; Content: ⁇ 99.0%; Trichloromethane: Grade: AR; Batch number: 20170410; Specification: 500
  • Chromatographic conditions temperature: 260°C; column flow: 2.0ml/min; purge flow: 20ml/min; split flow: 2ml/min; heating program: initial, hold at 80°C for 5 minutes, and increase to 230 at 10°C/min Hold for 25 minutes at °C; 45 minutes in total; Detector: Model: FID; Temperature: 280°C; Hydrogen: 40ml/min; Air: 450ml/min; Makeup gas: 45ml/min.
  • Dynamic light dispersion instrument model Litesizer 500; Manufacturer: Anton Paar (Shanghai) Trading Co., Ltd;
  • Sample Test the stock solution of sample solution.
  • Figure 1 is a dynamic light astigmatism spectrum of commercially available human serum albumin (Sample 1).
  • sample 2 Remove the 30nm-300nm particles in sample 1 according to the above method, and add 75 micrograms of poloxamer 188 (BASF batch number WPAK527B) per gram of human albumin to obtain sample 2.
  • the dynamic optical astigmatism spectrum of sample 2 is shown in the figure 2 shown.
  • poloxamer 188 can also be replaced with poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 or One of poloxamers 407, alternatively, poloxamer 188, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 or poloxamer 407 can also be used Many of them are used at the same time.
  • the inventors of the present application have also found through experiments that only a small amount of poloxamer can be added to achieve the desired effect.
  • 30nm-300nm is basically not detected. Small particles between 30nm-300nm, or only a few small particles between 30nm-300nm; when the amount of addition exceeds 500 micrograms of poloxamer/g human albumin, the microparticles between 30nm-300nm will be obvious increase.
  • human serum albumin (sample 3, Guangdong Weilun Biopharmaceutical Co., Ltd. batch number: 20170104), its prescription ingredients are: human serum albumin, and the excipients are sodium caprylate and sodium chloride.
  • the prescription complies with the requirements of the Pharmacopoeia, adding 0.159mmol of sodium caprylate per 1g of albumin without adding N-acetyltryptophan.
  • the pH is 6.7, and the total sodium content does not exceed 160mmol/L.
  • the method of Example 1 was used to test the residual long-chain fat content of sample 3, and the ratio of long-chain fatty acids (mol/mol human albumin): 0.34:1.
  • Figure 3 shows the dynamic optical astigmatism spectrum of sample 3.
  • sample 3 The 30nm-300nm particles in sample 3 were removed according to the method in Example 1, and 100 micrograms of poloxamer 188 (BASF batch number WPAK527B) was added to the processed human serum albumin per gram of human albumin to obtain sample 4 , Its dynamic optical astigmatism spectrum is shown in Figure 4.
  • Examples 1 and 2 show that the two standard human serum albumin prescriptions in the pharmacopoeia have particles of about 200 nm when poloxamer is not added; after adding a small amount of poloxamer, both can effectively inhibit and prevent micro The appearance of particles.
  • poloxamer 188 can also be replaced with poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 or One of poloxamers 407, alternatively, poloxamer 188, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 or poloxamer 407 can also be used Many of them are used at the same time.
  • the inventors of the present application have also found through experiments that only a small amount of poloxamer can be added to achieve the desired effect.
  • the detection of 30nm-300nm is basically not possible.
  • the high-purity recombinant human albumin stock solution obtained after improvement and purification is the ratio of long-chain fatty acids (mol/mol human Albumin) is equal to 0.059:1, which is nearly 10 times lower than the content of long-chain fatty acids derived from human serum. No long-chain fatty acids and poloxamers were added.
  • Sample 5 was prepared only in accordance with the requirements of the Chinese Pharmacopoeia 2015 version. Add 0.071mmol of sodium caprylate (Chengdu Huayi Pharmaceutical Excipient Manufacturing Co., Ltd.
  • Figure 5 is a dynamic light astigmatism spectrum of recombinant human albumin (sample 5) without poloxamer and long-chain fatty acids.
  • sample 6 In the high-purity recombinant human albumin stock solution obtained by the method of Example 3 above, sodium oleate (BBI Life Sciences batch number: E802BA0026) was added to the stock solution to the molar ratio of long-chain fatty acid/recombinant human albumin to 1.0 :1.0 (including the residual long-chain fatty acids in the stock solution), add 0.081mmol of sodium caprylate (Chengdu Huayi Pharmaceutical Excipient Manufacturing Co., Ltd.
  • Figure 6 is a dynamic light astigmatism chart of a recombinant human albumin-containing preparation (Sample 6) containing only long-chain fatty acids.
  • sample 7 In the high-purity recombinant human albumin stock solution obtained by the method of Example 3 above, add 100 micrograms of Poloxamer 188 (BASF batch number WPAK527B) per gram of human albumin, and add 0.079 mmol per 1g of albumin Sodium caprylate (Chengdu Hualux Pharmaceutical Excipient Manufacturing Co., Ltd. batch number 20170901), add 0.066mmol of acetyl tryptophan (Adamas Reagent Co., Ltd batch number: P230264) per 1g albumin, pH is 6.6, control the total sodium content More than 160mmol/L.
  • Poloxamer 188 BASF batch number WPAK527B
  • 0.079 mmol per 1g of albumin Sodium caprylate Choengdu Hualux Pharmaceutical Excipient Manufacturing Co., Ltd. batch number 20170901
  • acetyl tryptophan Adamas Reagent Co., Ltd batch number: P230264
  • Figure 7 is a dynamic light astigmatism chart of recombinant human albumin (sample 7) with only poloxamer added.
  • sample 8 In the high-purity recombinant human albumin stock solution obtained by the method of Example 3 above, 100 micrograms of poloxamer 188 (BASF batch number WPAK527B) was added per gram of human albumin, and sodium oleate (BBI) was added. Life Sciences batch number: E802BA0026) to the molar ratio of long-chain fatty acids/recombinant human albumin is 1.0:1.0 (including the residual long-chain fatty acids in the stock solution), and 0.083mmol of sodium caprylate (Chengdu Huayi Pharmaceutical Co., Ltd.) is added for every 1g of albumin. Excipient Manufacturing Co., Ltd.
  • Figure 8 is a dynamic light astigmatism spectrum of recombinant human albumin (sample 8) added with poloxamer and long-chain fatty acids.
  • Sample 9 In the high-purity recombinant human albumin stock solution obtained by the method of Example 3 above, 100 micrograms of poloxamer 188 (6BASF batch number WPAK527B) per gram of human albumin was added, and sodium soybean oleate was added (Qingdao Ruinuo Chemical Co., Ltd. batch number: 20190121) to the molar ratio of long-chain fatty acids/recombinant human albumin is 0.5:1.0 (including the residual long-chain fatty acids in the stock solution), and add 0.082mmol of sodium caprylate ( Chengdu Hualux Pharmaceutical Excipients Manufacturing Co., Ltd.
  • Figure 9 is a dynamic light astigmatism spectrum of recombinant human albumin (Sample 9) added with poloxamer and long-chain fatty acid (sodium soybean oleate).
  • sample 10 In the high-purity recombinant human albumin stock solution obtained by the method of Example 3 above, 50 micrograms of poloxamer 188 (BASF batch number WPAK527B) was added per gram of human albumin, and sodium stearate was added (Damas-beta batch number: P1042477) and sodium oleate (BBI Life Sciences batch number: E802BA0026) 1: 2 moles after mixing uniformly, the molar ratio of mixed long-chain fatty acid/recombinant human albumin is 0.6: 1.0 (the stock solution has residual long-chain Fatty acids included), and 0.086mmol of sodium caprylate per 1g of albumin (Chengdu Hualux Pharmaceutical Excipients Manufacturing Co., Ltd.
  • Figure 10 is a dynamic light astigmatism spectrum of recombinant human albumin (sample 10) added with poloxamer and long-chain fatty acids (sodium stearate/sodium oleate mixture).
  • sample 11 In the high-purity recombinant human albumin stock solution obtained by the method of Example 3 above, 50 micrograms of poloxamer 188 (BASF batch number WPAK527B) was added per gram of human albumin, and sodium stearate was added (Damas-beta batch number: P1042477) and sodium oleate (BBI Life Sciences batch number: E802BA0026) 1: 2 moles after mixing uniformly, the molar ratio of mixed long-chain fatty acid/recombinant human albumin is 0.6: 1.0 (the stock solution has residual long-chain Fatty acids are included), and 0.153mmol of sodium caprylate (Chengdu Huayi Pharmaceutical Excipients Manufacturing Co., Ltd. batch number 20170901) is added per 1g of albumin, the pH is 7.0, and the total sodium content is controlled not to exceed 160mmol/L.
  • poloxamer 188 BASF batch number WPAK527B
  • Figure 11 is a dynamic light astigmatism spectrum of recombinant human albumin (Sample 11) added with poloxamer and long-chain fatty acid (sodium stearate/sodium oleate mixture).
  • the long-chain fatty acid can also be myristic acid (C14:0), palmitic acid (C16:0), stearic acid (C18:0), oil One or more of acid (C18:1), linoleic acid (C18:2), linolenic acid (C18:3), and arachidonic acid (C20:4). Tests have shown that these long-chain fatty acids can also achieve the same effect, that is, when combined with poloxamer, they can promote the stability of recombinant human albumin preparations. It was negative in the above stability test.
  • poloxamer 188 can also be replaced with poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 or One of poloxamers 407, alternatively, poloxamer 188, poloxamer 124, poloxamer 188, poloxamer 237, poloxamer 338 or poloxamer 407 can also be used Many of them are used simultaneously. Tests have proved that these poloxamers can also achieve the same effect. After adding an appropriate amount of long-chain fatty acids, the stability of human serum albumin with a small amount of poloxamer can meet the requirements.
  • the inventor of the present application also found through experiments that only a small amount of poloxamer can be added to achieve the desired effect. Generally, when added to more than 10 micrograms of poloxamer/g human albumin, the stability observation results are basically negative. ; When the addition amount exceeds 500 micrograms poloxamer/g human albumin, the stability observation results begin to appear positive.
  • the inventor of the present application also found through experiments that when the molar ratio of long-chain fatty acids to human albumin is 0.2-3.0, especially 0.3-2.0, the stability of the human albumin preparation is in the best state.
  • the stability observation results start to be positive in a small amount; when the molar ratio of long-chain fatty acid to human albumin is lower than 0.2 or higher than 3.0, it is stable Sexual observations began to appear positive in large numbers.
  • the samples 5-11 prepared by the method of Example 3-9 were subjected to differential thermal scanning analysis (DSC) spectra.
  • DSC model Nano DSC 602000; manufacturer: TA; sample diluent: 0.01mol/L phosphoric acid, neutral pH.
  • Experimental conditions Dilute the original sample solution to 1-5 mg/ml with a diluent; heating range: 45°C-90°C; step size: 1°C/min. The experimental results are shown in Table 2.
  • the highly purified recombinant human albumin is almost completely defatted, and poloxamer needs to be added to inhibit the appearance of 30nm-300nm particles.
  • the results of comparative experiments show that only poloxamer, sodium caprylate (medium chain fatty acid) and acetyl tryptophan in the conventional pharmacopoeia are added.
  • medium chain fatty acids can effectively increase Tonset , T m1 and T m2 , they lack long-chain fatty acid dialogue.
  • the ligands of each adjacent domain of the protein support each other and still cannot meet the requirements of thermal stability.
  • controlling the content of long-chain fatty acids can effectively increase Tonset , T m1 and T m2 , and increase the relative stability of protein domains while adding a small amount of poloxamer to inhibit hydrophobic interactions.
  • the human albumin-containing preparation of the present invention can meet the stability requirements of 57°C/50h incubation, 30°C/14 days incubation and 20°C/28 days incubation. Its stability index is excellent, and it is suitable for long-term 2°C-8°C and room temperature. Stability requirements for storage and transportation.

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Abstract

一种制备人白蛋白的制剂的方法，包括控制制剂中长链脂肪酸的含量以及添加泊洛沙姆。长链脂肪酸选自肉豆蔻酸、棕榈酸、硬脂酸、油酸、亚油酸、亚麻酸、花生四烯酸的一种或多种，其与人白蛋白的摩尔比为0.2-3.0。泊洛沙姆的含量为10-500微克/克人白蛋白。由所述方法制备的人白蛋白制剂具有提高的稳定性，不含由人白蛋白团聚和/或原纤维化形成的粒径在30nm以上的颗粒。

Description

含人白蛋白的制剂及其制备方法 技术领域
本发明涉及生物医药领域，尤其涉及一种含人白蛋白的制剂及其制备方法。在本申请中，术语“人白蛋白”包括人血清白蛋白(Human Serum Albumin)、重组人白蛋白(Recombinant Human Albumin)。重组人白蛋白也称为重组人血清白蛋白(Recombinant Human Serum Albumin)，或者是重组人白蛋白突变体(Recombinant Human Albumin Mutant)。
背景技术
人白蛋白主要的药理作用，包括调节组织与血管间水分的动态平衡，维持正常与恒定的血浆容量，同时还对某些离子和化合物有较高亲和力，与这些物质可逆结合，发挥转运功能。人白蛋白还为肌体提供了大量的氨基酸储备。由于人白蛋白的上述作用，使其可以应用于临床各学科，发挥多种治疗作用。人白蛋白临床主要用于调节血浆胶体渗透压、扩充血容量，治疗创伤性、出血性休克，严重烧伤及低蛋白血症，在中风，肝硬化肝腹水和肾病等常见疾病中都有着广泛的应用。除了直接在临床治疗领域，白蛋白还在疫苗生产所使用的培养基、药品辅料、诊断试剂、新型肿瘤长效制剂产品、化妆品、实验室生物试剂等多个方面，都已经得到了极其广泛的应用。
人白蛋白的结构是一个心型结构的单链非糖基化蛋白，有585个氨基酸，17对二硫键，一个自由巯基，分子量为66438道尔顿。人白蛋白在人体中的半衰期为19～21天。人白蛋白的心形结构由三个主要结构域和六个被17个二硫键包裹的亚结构域组成，它们通过范德华力松散地结合在一起。从其晶体结构可以看出，二硫化物桥赋予螺旋状的球状结构刚度，但提供足够的灵活性，使蛋白质能够根据周围介质的变化进行构象变化。
人白蛋白常规的生产方法是从人血清中分离纯化提取纯化出来，统称为人血清白蛋白。人血清白蛋白的发现和使用已历经几十年，发展成熟为一套完整的生产和制剂工艺(Cohn法-超滤-/或纯化-巴氏灭活-制剂(辛酸钠和/或N-乙酰色氨酸))，其制剂处方已经成为全球各国药典的通用标准。人血清来源的白蛋白稳定性良好，药典中要求其制剂成品置57℃±0.5℃水浴中保温50小时，除颜色有轻微变化外，应无肉眼可见的其他变化。血清来源的白蛋白制剂全部成品应置20-25℃至少4周或30-32℃至少14天，出现浑浊或烟雾状沉淀之瓶进行相关的检查。即使对人血清白蛋白进行如此严格的热稳定检查，临床中依然出现与微小团聚体(aggregation)和原纤维化(fibrillation)聚集体蛋白相关的静脉炎。临床研究证明采用15微米孔径的普通输液器进行静脉输注 人血清白蛋白，患者静脉炎的发生率为20％；而采用5微米孔径的精密输液器进行输注人血清白蛋白，患者静脉炎的发生率仅为2.5％。由此证明，人血清白蛋白生产工艺中采用了0.1微米～0.22微米的除菌和除颗粒的过程后，制剂在运输和存放中依然出现新的微小团聚体和微小原纤维化蛋白，导致患者出现较大比例的白蛋白输注静脉炎(薛小萍等临床护理杂志2010年10月第9卷第5期第69～70页)。因此，在一些人血清白蛋白的生产厂家的说明书中明确了使用精密输液器的要求。即使如此，上述的部分不稳定白蛋白的粒径在30nm-100nm之间，一般的除菌滤膜(0.1微米～0.22微米)不能有效除去。该直径的聚集体介于多聚体和可见异物之间，是产生免疫原性等不良反应的主要原因。
人白蛋白的另外一个来源是通过基因重组的方法，培养能够特异性表达人白蛋白的宿主，比如真菌类真核单细胞的毕赤酵母或酿酒酵母等表达的重组人白蛋白，或者是水稻转基因表达的重组人白蛋白。重组人白蛋白经高度纯化后的成品，采用与药典规定的相同的制剂要求，其稳定性远远达不到人血清来源的人白蛋白。为此，申请号为CN201610017874.7的中国专利申请公开了采用在药典标准处方的基础上添加吐温80或吐温20方法阻止重组人白蛋白聚合物的产生。
S.Baldursdottir等人公开了重组人白蛋白溶液中赋形剂聚山梨醇酯80(吐温80)的稳定作用，并观察，辅料辛酸盐和在recombumin 
Figure PCTCN2020086057-appb-000001
配方中使用，这两种辅料都增加了蛋白质的稳定性，辛酸盐在较高温度下似乎能有效地保护蛋白质，而聚山梨酸盐(吐温)在较低温度下则能防止自结合。此外，还观察到辛酸盐和聚山梨酯80的非线性协同作用。(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.)
聚山梨醇酯的负面影响是非离子表面活性剂中残留的烷基氧化物催化蛋白质氧化的可能增加。例如，重组人纤毛神经营养因子(CNTF)被聚山梨醇酯80中的烷基过氧化物二聚化。聚山梨醇酯80中的过氧化物水平与重组人粒细胞集落刺激因子(rhG-CSF)中的氧化程度之间存在相关性，并且过氧化物诱导的氧化似乎比小瓶顶部空间中的大气氧诱导的氧化更严重(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和Herman AC,Boone TC,Lu HS.1996.Characterization,formulation,and stability of Neupogen(Filgrastim),a recombinant human granulocyte-colony stimulating factor.Pharm.Biotechnol.9:303–328.)。
人白蛋白中有蛋氨酸和一个自由巯基，在体内起到抵抗自由基的过氧化作用，防止细胞和器官受到伤害。因此，聚山梨醇酯的质量和储存条件需要得到很好的控制， 并且它们在蛋白质中的使用量相关性需要保持在最低水平(AGGREGATION OF THERAPEUTIC PROTEINS，P341,Edited by Wei Wang，Christopher J.Roberts，WILEY2010)，否则人白蛋白将被部分氧化失去应有的氧化还原生理活性，同时氧化的人白蛋白将进一步促使白蛋白去折叠，加速人白蛋白的团聚和纤维化。
人白蛋白在体内循环中，具有一个非常重要的生理作用是依赖配体结合特性运载药物、脂肪酸、维生素、细胞色素和金属离子等作用。
脂肪酸作为人白蛋白的配体，对人白蛋白的结构和热稳定性有决定性的作用。人血清白蛋白中添加辛酸钠作为配体稳定剂，可以有效地提高热焓值和T m(熔化温度)值，从而增加热稳定性。
Brian等人公开了使用差式扫描量热法(DSC)来确定从混浆的血液、转基因酵母和转基因水稻制备的人白蛋白的市售产品的蛋白去折叠特性。添加油酸后，人白蛋白产品去折叠转变的初始熔化温度(T m1)从62℃升高到75℃不等。该研究证明了脂肪酸配体对人白蛋白热稳定性的重要性，但是它没有研究因蛋白质疏水区暴露导致的团聚和原纤维化，同时DSC也不能表征出低温和常温以及运输震动条件下的团聚和原纤维化。(Brian E.Lang and Kenneth D.Cole，Biotechnol.Prog.,2014,Unfolding Properties of Recombinant Human Serum Albumin Products Are Due To Bioprocessing Steps，DOI10.1002/btpr.1996)。
除了脂肪酸以外，人血清来源的白蛋白还有胆红素、激素、脂溶性药物、血红色等配体与白蛋白紧密结合，增加了白蛋白的构型稳定性。当人血清来源的白蛋白被脱脂后，其稳定性急剧下降，与高度纯化的重组人白蛋白完全一致。
吐温等表面活性剂作为稳定剂应尽可能减少其添加量，以避免其毒性作用，更为重要的是减少吐温等表面活性剂对人白蛋白配体生理作用的干扰，或者寻找更为安全的和更低剂量的表面活性剂。
泊洛沙姆(poloxamer)为聚氧乙烯-聚氧丙烯共聚物的通用名称。德国BASF公司生产的商品名为普朗尼克(Pluronic)，属于非离子型表面活性剂。泊洛沙姆是目前使用在静脉乳剂中唯一合成的乳化剂，其中泊洛沙姆188具有最佳乳化性能和安全性，是一种O/W型乳化剂，常用浓度为0.3％(王猛等，药学与临床研究2007年第15卷第1期第10～13页)。
作为非离子型表面活性剂，泊洛沙姆188可以通过形成胶束增加难溶性药物的溶解度，其临界胶束浓度(CMC)为0.6％左右。泊洛沙姆188可以抑制血栓的形成、影响血液流变学的活动，细胞膜密封，吞噬细胞的活化(刺激的吞噬功能和超负离子产生)和中性粒细胞脱颗粒、预防成年人骨骼肌坏死。泊洛沙姆188还可以治疗脑损伤：研究表明泊洛沙姆188可以减少大鼠由于试验性脑损伤所引起的炎症及组织损伤，在其他试验模型中也有证据表明其可以对抗组织损伤。其保护机制可能涉及表面活性剂对氧化的应激性和炎症反应的影响。此外泊洛沙姆188还可以提高细胞存活率。研究 证明静脉注射单一剂量的泊洛沙姆188可以通过建立完整的循环从而有效的恢复受损的组织。泊洛沙姆188可以稳定由于多种原因引起的细胞膜缺陷。大鼠的研究表明单剂量的泊洛沙姆188可以对抗出血后早期神经元损伤，但对长期出血所导致的神经元损伤没有效果，但是每天给予泊洛沙姆188可能可以产生长期、有效的神经元保护作用。
申请号为CN200480003079.8的中国专利申请公开了泊洛沙姆作为表面活性剂稳定造血因子、甲状旁腺激素或抗体，该专利中的蛋白质没有脂肪酸的配体作用，因而其添加了较大剂量的泊洛沙姆，以稳定蛋白质和避免吐温的氧化损伤。因造血因子、甲状旁腺激素或抗体是小容量注射剂(ug～mg/瓶)，即使是较大量浓度的泊洛沙姆添加，其泊洛沙姆的总量并不大。然而，人白蛋白是大容量注射剂，注射剂量在5～12.5g/瓶，即使是少量浓度的吐温或泊洛沙姆添加，其吐温或泊洛沙姆的总量就较大。
因此，本领域的技术人员致力于研究一种含人白蛋白的制剂或其制备方法以抑制人白蛋白形成团聚物和原纤维化微小颗粒，增加人白蛋白临床使用的安全性和耐受性，同时，需要避免公开处方中吐温的毒性和吐温氧化物导致的蛋白质氧化变性。
发明内容
本发明所要解决的技术问题是增加人白蛋白制剂的稳定性。
为解决所要解决的技术问题，本发明提供了一种制备含人白蛋白的制剂的方法，所述方法包括：通过控制所述制剂中的长链脂肪酸的含量以及添加泊洛沙姆来提高所述制剂中的人白蛋白的稳定性。
优选地，所述方法还包括：通过减少人白蛋白的团聚和原纤维化来提高所述制剂中的人白蛋白的稳定性。
优选地，所述方法还包括：提高所述制剂中的人白蛋白的稳定性使得所述制剂制备完成后不出现由人白蛋白团聚和/或原纤维化形成的粒径在30nm以上的颗粒。
优选地，所述方法还包括：提高所述制剂中的人白蛋白的稳定性使得所述制剂制备完成后不出现由人白蛋白团聚和/或原纤维化形成的粒径在100nm以上的颗粒。
优选地，所述方法还包括：提高所述制剂中的人白蛋白的稳定性使得所述制剂制备完成后不出现由人白蛋白团聚和/或原纤维化形成的粒径在200nm左右的颗粒。
优选地，所述方法还包括：提高所述制剂中的人白蛋白的稳定性使得稳定性达到当地药典的要求。
优选地，所述方法还包括：提高所述制剂中的人白蛋白的稳定性使得稳定性达到如下条件：在57℃培育50h、30℃培育14天和20℃培育28天中不出现浑浊和可见异物。
优选地，所述人白蛋白为人血清白蛋白，所述方法包括：通过添加泊洛沙姆来提高所述制剂中的人白蛋白的稳定性。
优选地，所述人白蛋白为重组人白蛋白，所述方法包括：通过添加长链脂肪酸和泊洛沙姆来提高所述制剂中的人白蛋白的稳定性。
优选地，所述长链脂肪酸以盐的方式添加。
优选地，所述长链脂肪酸以钠盐的方式添加。
优选地，所述方法还包括：所述制剂制备完成后，所述长链脂肪酸与人白蛋白的摩尔比为0.2-3.0。
优选地，所述方法还包括：所述制剂制备完成后，所述长链脂肪酸与人白蛋白的摩尔比为0.3-2.0。
优选地，所述方法还包括：所述制剂制备完成后，所述泊洛沙姆的含量为10-500微克/克人白蛋白。
优选地，所述泊洛沙姆选自泊洛沙姆124、泊洛沙姆188、泊洛沙姆237、泊洛沙姆338和泊洛沙姆407中的一种或多种。
优选地，所述泊洛沙姆是泊洛沙姆188。
优选地，所述长链脂肪酸选自肉豆蔻酸(C14:0)、棕榈酸(C16:0)、硬脂酸(C18:0)、油酸(C18:1)、亚油酸(C18:2)、亚麻酸(C18:3)、花生四烯酸酸(C20:4)中的一种或多种。
优选地，所述长链脂肪酸为大豆油酸或全氢化大豆油酸。
优选地，所述方法还包括：添加当地药典所要求的成分以及含量。
优选地，当地药典所要求的成分包括辛酸钠或/和N-乙酰色氨酸。
本发明还提供了根据本发明所提供的方法所制备的含人白蛋白的制剂。
本发明还提供了一种含人白蛋白的制剂，所述制剂不含有由人白蛋白团聚和/或原纤维化形成的粒径在30nm以上的颗粒。
本发明还提供了一种含人白蛋白的制剂，所述制剂不含有由人白蛋白团聚和/或原纤维化形成的粒径在100nm以上的颗粒。
本发明还提供了一种含人白蛋白的制剂，所述制剂不含有由人白蛋白团聚和/或原纤维化形成的粒径在200nm左右的颗粒。
本发明还提供了一种含人白蛋白的制剂，所述方法还包括：所述制剂中的人白蛋白的稳定性达到如下条件：在57℃培育50h、30℃培育14天和20℃培育28天中不出现浑浊和可见异物。
本发明还提供了一种含人白蛋白的制剂，所述制剂中含有泊洛沙姆和长链脂肪酸。
优选地，所述制剂中的长链脂肪酸与人白蛋白的摩尔比为0.2-3.0。
优选地，所述制剂中的长链脂肪酸与人白蛋白的摩尔比为0.3-2.0。
优选地，所述方法还包括：所述制剂中的泊洛沙姆的含量为10-500微克/克人白蛋白。
优选地，所述泊洛沙姆选自泊洛沙姆124、泊洛沙姆188、泊洛沙姆237、泊洛沙 姆338和泊洛沙姆407中的一种或多种。
优选地，所述泊洛沙姆是泊洛沙姆188。
优选地，所述长链脂肪酸选自肉豆蔻酸(C14:0)、棕榈酸(C16:0)、硬脂酸(C18:0)、油酸(C18:1)、亚油酸(C18:2)、亚麻酸(C18:3)、花生四烯酸酸(C20:4)中的一种或多种。
优选地，所述长链脂肪酸为大豆油酸或全氢化大豆油酸。
优选地，所述制剂还含有当地药典所要求的成分以及含量。
优选地，当地药典所要求的成分包括辛酸钠或/和N-乙酰色氨酸。
本发明是依据脂肪酸配体增加人白蛋白的热变性稳定性，泊洛沙姆作为疏水补丁屏蔽疏水力相互作用和界面疏水相互作用减少团聚和原纤维化，从而提高低温、常温、高温下或者震荡运输过程中的团聚和原纤维化导致的稳定性。这种团聚物/聚集体和原纤维化颗粒的直径在30nm-300nm之间，主要在100-200nm之间，HPLC-SEC检测不出，0.1μm～0.2μm的膜系统不能有效过滤，更为重要的是不稳定的白蛋白溶液体系(即使去除干净该团聚物/聚集体和原纤维化颗粒)在存放和运输过程中会不断产生，并增加直径，直至肉眼可见到微米和亚毫米的直径。
为了实现各种条件下抑制人白蛋白的团聚和原纤维化，本申请的发明人进行了极为细致的研究，对人血来源的白蛋白进行剖析，结果发现人血来源的白蛋白中每摩尔含有0.3到0.8mol的长链脂肪酸。而人体中循环的人白蛋白每摩尔含有0.5到2.5mol的长链脂肪酸，以起到脂肪酸动员和存储的生理功能。在人血来源的白蛋白混浆生产纯化工艺中失去了一部分长链脂肪酸。按照中国药典2015版第三部，在巴氏灭活和制剂中每1g白蛋白添加0.140～0.180mmol的辛酸钠；或者与乙酰色氨酸混用，每1g白蛋白添加0.064～0.096mmol的辛酸钠，每1g白蛋白添加0.064～0.096mmol的乙酰色氨酸。同时作为大容量注射剂，制剂应满足人体的生理条件，pH为6.4～7.4，渗透压摩尔浓度为210～400mOsmol/kg。
无论哪种方式表达的重组人白蛋白，都需要进行多步骤的分离、层析和超滤透析等纯化工艺，在彻底去除宿主蛋白、多糖、内毒素和各种污染的同时，也将脂肪酸配体去除干净，成为脱脂的人白蛋白。所以，失去脂肪酸和其他人体内的各种配体后的重组人白蛋白的稳定性比人血源的白蛋白要低得多。因此，适当添加一定量的表面活性剂，但是其浓度低于CMC的浓度，其表面活性剂的作用就是起到失去配体的白蛋白的疏水补丁，以阻止因白蛋白/空气/容器/震荡气泡等各种疏水界面相互作用导致的去折叠后的团聚作用。
因此，本发明在中国药典、美国药典、欧洲药典等药典的制剂通则和人血清白蛋白的基础上，加入一定量泊洛沙姆和调节长链脂肪酸的比例，使得人白蛋白在各国药典规定的条件下满足稳定性的要求。
本发明的优势在于可以直接添加长链脂肪酸配体到人体循环的合理比例与少量的 泊洛沙姆，或利用已有的人白蛋白常规处方，再添加少量的泊洛沙姆，使得人白蛋白的热稳定性和存储、运输过程中不产生团聚/聚集(Aggregation)和原纤维化(Fibrillation)。这种团聚物/聚集体和原纤维化的直径在50nm～300nm之间，主要在100～200nm之间，HPLC-SEC检测不出，0.1μm～0.2μm的膜系统不能有效过滤，更为重要的是不稳定的白蛋白溶液体系(即使去除干净该团聚物)在存放和运输过程中会不断产生，并增加直径，直至肉眼可见到微米和亚毫米的直径。
以下将结合附图对本发明的构思、具体结构及产生的技术效果作进一步说明，以充分地了解本发明的目的、特征和效果。
附图说明
图1为实施例1中样品1(含有辛酸钠和乙酰色氨酸的人血清白蛋白)的动态光散色仪(Dynamic Light Scattering简写DLS)图谱。
图2为实施例1中样品2(含有辛酸钠和乙酰色氨酸的人血清白蛋白，添加泊洛沙姆)的DLS图谱。
图3为实施例2中样品3(市售的仅含有辛酸钠的人血清白蛋白)的DLS图谱。
图4为实施例2中样品4(市售的仅含有辛酸钠，添加泊洛沙姆后的人血清白蛋白)的DLS图谱。
图5为实施例3中样品5(含有辛酸钠和乙酰色氨酸，未添加泊洛沙姆和长链脂肪酸的重组人白蛋白)的DLS图谱。
图6为实施例4中样品6(含有辛酸钠和乙酰色氨酸，仅添加长链脂肪酸(油酸)的重组人白蛋白)的DLS图谱。。
图7为实施例5中样品7(含有辛酸钠和乙酰色氨酸，仅添加泊洛沙姆的重组人白蛋白)的DLS图谱。
图8为实施例6中样品8(含有辛酸钠和乙酰色氨酸，添加泊洛沙姆和长链脂肪酸(油酸)的重组人白蛋白)的DLS图谱。
图9为实施例7中样品9(含有辛酸钠和乙酰色氨酸，添加泊洛沙姆和长链脂肪酸(大豆油酸钠)的重组人白蛋白)的DLS图谱。
图10为实施例8中样品10(含有辛酸钠和乙酰色氨酸，添加泊洛沙姆和长链脂肪酸(硬脂酸钠/油酸钠混合)的重组人白蛋白)的DLS图谱。
图11为实施例9中样品11(含有辛酸钠，添加泊洛沙姆和添加长链脂肪酸(硬脂酸钠/油酸钠混合)的重组人白蛋白)的DLS图谱。
具体实施方式
以下通过特定的具体实例说明本发明的实施方式，本领域技术人员可由本说明书所揭露的内容轻易地了解本发明的其他优点与功效。本发明还可以通过另外不同的具 体实施方式加以实施或应用，本说明书中的各项细节也可以基于不同观点与应用，在没有背离本发明的精神下进行各种修饰或改变。需说明的是，在不冲突的情况下，以下实施例及实施例中的特征可以相互组合。
以下对本申请所使用的术语进行定义和解释。
术语“人白蛋白”指人血清白蛋白/或重组人白蛋白，重组人白蛋白也称为重组人血清白蛋白，或者是重组人白蛋白突变体。重组人白蛋白至少包括微生物基因重组的人白蛋白、真核细胞重组人白蛋白、植物转基因的重组人白蛋白和动物转基因重组人白蛋白等。
术语“制剂”应当被理解为至少包括溶液制剂、冷冻干燥制剂或低温喷雾干燥制剂，优选溶液制剂，溶液制剂通常被无菌灌装在确定容积的玻璃或塑料容器中。
术语“含人白蛋白的制剂”应当被理解为适用于临床注射、培养基、药用辅料、医疗器械、疫苗辅料、美容、分析诊断试剂等多个方面的不同的人白蛋白浓度的制剂，例如人白蛋白浓度1％～30％，优选10％～25％。
泊洛沙姆是非离子表面活性剂，是环氧乙烷和1，2-环氧丙烷的嵌段共聚物，其通式为H(C2H4O) a(C3H6O) b(C2H4O) aOH。泊洛沙姆188在中国药典2015年版、美国药典、欧洲药典中和已经批准的药物中被列入静脉注射的脂肪乳剂的乳化剂，抗体注射剂的稳定剂，其生物安全度高。
术语“长链脂肪酸”是指碳链上碳原子数大于12的脂肪酸，应当理解为人体可接受的各种来源(合成或提取)的脂肪酸，或以各种形式存在的脂肪酸(例如，盐，尤其是钠盐)，包括但不限于肉豆蔻酸(C14:0)、棕榈酸(C16:0)、硬脂酸(C18:0)、油酸(C18:1)、亚油酸(C18:2)、亚麻酸(C18:3)和花生四烯酸酸(C20:4)中的一种或多种。
术语“控制”的主体是含人白蛋白的制剂中的长链脂肪酸的含量，是指将含人白蛋白的制剂中的长链脂肪酸的量维持在给定范围内：当现有方法制得的含人白蛋白的制剂中本身含有长链脂肪酸，例如人血来源的含人白蛋白的制剂，如果长链脂肪酸含量低于给定范围内，“控制”是指在制备含人白蛋白的制剂的过程中添加长链脂肪酸使得最终制得的含人白蛋白的制剂中的长链脂肪酸在给定范围内，如果长链脂肪酸已经在给定范围内，“控制”则指在制备含人白蛋白的制剂的过程中无需任何操作；当现有方法制得的含人白蛋白的制剂不含有或含有极少量的长链脂肪酸时，“控制”是指在制备含人白蛋白的制剂的过程中添加长链脂肪酸使得最终制得的含人白蛋白的制剂中的长链脂肪酸在给定范围内。
术语“长链脂肪酸”应当被理解为一个固定词组，其中的“或”与这个词组的上下文不存在任何逻辑关系，在计算“长链脂肪酸”的含量时是指长链脂肪酸的量总和，为使得长链脂肪酸的含量在给定范围内，可以加入长链脂肪酸，也可以加入长链脂肪酸的混合物。
术语“长链脂肪酸的含量”应当理解为最终制得的含人白蛋白的制剂中长链脂肪酸的含量，包括由于原料本身含有长链脂肪酸而在制备过程中残留的长链脂肪酸的量和额外添加(如果在“控制”过程中有添加的话)的长链脂肪酸的量。
术语“添加”，当其主体是泊洛沙姆时，应当理解为在制备含人白蛋白的制剂的过程中加入泊洛沙姆使得最终制得的含人白蛋白的制剂中的泊洛沙姆在给定范围内。
“控制”和“添加”的步骤应当理解为在制备含人白蛋白的制剂的过程中实施，使得最终制得的含人白蛋白的制剂中长链脂肪酸的含量以及泊洛沙姆的含量均在给定范围内，例如可以在制剂配制的半成品原液实施，也可以通过超滤和透析过程实施，也可以在巴氏灭活前作为蛋白稳定剂添加，还可以在纯化层析工艺的最后一步到最后几步中实施，目的是为了实现最终制得的含人白蛋白的制剂获得本发明规定的泊洛沙姆和长链脂肪酸的含量范围。
术语“提高”应当理解为本发明的含人白蛋白的制剂的稳定性高于现有的人白蛋白的稳定性，例如本发明实现了含人白蛋白的制剂在储存、运输、常温培育过程中不出现由人白蛋白团聚/聚集或原纤维化等形成的粒径为30nm-300nm的颗粒。
术语“颗粒”应当被理解为含人白蛋白的制剂在储存、运输、常温培育等过程中出现的由人白蛋白团聚/聚集或原纤维化而形成的具有一定粒径范围的物质，本发明优选粒径范围30nm-300nm。
本发明的含人白蛋白的制剂可以用于抑制冷干、冻干和低温喷雾干燥的冻融形成的聚合体、团聚物和纤维化，加快复溶。
本发明的含人白蛋白的制剂包括但不限于采用Cohn法和层析精纯制造的人血清来源的白蛋白、经不同步骤纯化的重组表达的人白蛋白。
本发明的含人白蛋白的制剂的pH值范围为6-8，优选各国药典规范的pH值6.4～7.4。
本发明的含人白蛋白的制剂中还可以包括稀释剂、缓冲剂、增溶剂、赋形剂、pH调节剂、含硫还原剂、抗氧化剂等。含硫还原剂包括N-乙酰DL-色氨酸(N-乙酰色氨酸)、N-乙酰-L-色氨酸，N-乙酰蛋氨酸、N-乙酰-L-蛋氨酸等。常用的缓冲剂和稀释剂包括氯化钠溶液、磷酸钠缓冲溶液、醋酸钠溶液、柠檬酸钠溶液，优选各国药典规范的范围。
本发明的含人白蛋白的制剂中还可以添加中链脂肪酸，例如辛酸钠、庚酸钠、癸酸钠，优选药典规范的辛酸钠。
本发明的含人白蛋白的制剂包括但不限于应用在临床注射、培养基、药用辅料、医疗器械、疫苗辅料、美容、分析诊断试剂等多个方面。
以下通过特定的具体实例说明本发明的实施方式。
实施例1
市售人血清白蛋白(样品1，上海莱士血液制品股份有限公司，批号：201708A010)，其处方为：人血白蛋白，辅料为辛酸钠，乙酰色氨酸。其处方符合药典要求，为每1g白蛋白添加0.079mmol的辛酸钠，每1g白蛋白添加0.076mmol的乙酰色氨酸，pH为6.8。采用气相色谱方法测试血清来源的人白蛋白经纯化后残留的长链脂肪含量。气相色谱分析人白蛋白的具体方法如下：
气相色谱仪：型号：7890A；厂家：安捷伦科技有限公司；
色谱柱：酸改性聚乙二醇毛细管柱TG-WAXMS A赛默飞科技有限公司；规格：30m*0.32mm*0.25μm；
材料和试剂：C17烷酸：级别：USA标准物质；批号：N-17A-JY16X；规格：＞100mg；含量：≥99.0％；棕榈酸：级别：国家标准物质；批号：190032-201603；规格：200mg/支；硬脂酸：级别：国家标准物质；批号：190032-201603；规格：200mg/支；油酸：级别：国家标准物质；批号：111621-2015-06；规格：100mg/支；含量：≥99.0％；亚油酸：级别：USA标准物质；批号：U-59A-J6-Y；规格：＞100mg/支；含量：≥99.0％；亚麻油酸：级别：USA标准物质；批号：U-62A-S20-B；规格：＞100mg/支；含量：≥99.0％；三氯甲烷：级别：AR；批号：20170410；规格：500ml/瓶；含量：≥99.0％；厂家：国药。
实验方法：按照《中华人民共和国药典》2015年版：通过加入内标物质C17烷酸酸，计算棕榈酸、硬脂酸、油酸、亚油酸、亚麻油酸与C17烷酸绝对峰面积，以各脂肪酸对照品溶液峰面积与内标峰面积比对各脂肪酸对照品溶液量作直线回归，求得直线回归方程，计算出供试品溶液各脂肪酸绝对峰面积，从而计算出供试品各脂肪酸含量。
色谱条件：温度：260℃；柱流量：2.0ml/min；吹扫流量：20ml/min；分流流量：2ml/min；升温程序：初始，80℃保持5分钟，以10℃/min升到230℃保持25分钟；共计45分钟；检测器：型号：FID；温度：280℃；氢气：40ml/min；空气：450ml/min；尾吹气：45ml/min。
精密称取C17烷酸制备内标溶液；精密称取脂肪酸标准品混合为对照品溶液。测试出标准曲线，可计算出人白蛋白中脂肪酸的含量。
人血白蛋白注射液(上海莱士201703A010)的脂肪酸含量检测结果：
C16:0(棕榈酸)含量(mol/mol蛋白)：0.241
C18:0(硬脂酸)含量(mol/mol蛋白)：0.147
C18:1(油酸)(mol/mol蛋白)：0.077
C18:2(亚油酸)(mol/mol蛋白)：0.085
C18:3亚麻油酸(mol/mol蛋白)：未检出
C20:4(花生四烯酸)(mol/mol蛋白)：未检出
以上合计：长链脂肪酸比例(mol/mol人白蛋白)：0.55：1
采用动态光散色仪测试，
动态光散色仪型号：Litesizer 500；厂家：Anton Paar(Shanghai)Trading Co.,Ltd；
比色皿：石英样品池；
样品：样品溶液原液测试。
实验方法：测量角度：自动；升温范围：25℃-60℃；步长：5℃
市售人血清白蛋白中不稳定性导致的30nm-300nm颗粒，需通过分子筛层析去除；其色谱条件：G-25填料(GE通用电气公司)，层析设备：EV 50D层析系统(苏州利穗科技)，缓冲液体系：0.1mol/L磷酸溶液，pH中性，纯化水；样品层析后采用30K超滤膜进行浓缩，按照原制剂处方配制成制剂进行测试。
图1为市售人血清白蛋白(样品1)的动态光散色仪图谱。
按照上述方法去除样品1中的30nm-300nm颗粒，并按照每克人白蛋白中添加75微克泊洛沙姆188(BASF批号WPAK527B)以得到样品2，样品2的动态光散色仪图谱如图2所示。
图1和图2的动态光散色仪的结果表明，在25℃和60℃两个温度点上，未添加泊洛沙姆的市售人血清白蛋白(样品1)在30nm-300nm之间出现微小颗粒，而除去颗粒后，少量添加泊洛沙姆的人血清白蛋白(样品2)在25℃和60℃两个温度点上都未出现微小颗粒。
本申请发明人通过试验还发现，作为该具体实施例的变形，泊洛沙姆188也可以替换成泊洛沙姆124、泊洛沙姆188、泊洛沙姆237、泊洛沙姆338或泊洛沙姆407中的一种，或者，也可以采用泊洛沙姆188、泊洛沙姆124、泊洛沙姆188、泊洛沙姆237、泊洛沙姆338或泊洛沙姆407中的多种同时使用。试验证明，这些泊洛沙姆也可以实现同样的效果，即除去颗粒后，少量添加泊洛沙姆的人血清白蛋白在25℃和60℃两个温度点上都未出现30nm-300nm之间的微小颗粒。
本申请发明人通过试验还发现，只需要添加少量的泊洛沙姆即可以达到理想的效果，通常，添加到10微克泊洛沙姆/克人白蛋白以上，基本上检测不到30nm-300nm之间的微小颗粒，或者，只出现很少的30nm-300nm之间的微小颗粒；添加量超过500微克泊洛沙姆/克人白蛋白时，30nm-300nm之间的微小颗粒会有明显的增多。
实施例2
市售人血清白蛋白(样品3，广东卫伦生物制药有限公司批号：20170104)，其处方成分为：人血白蛋白，辅料为辛酸钠，氯化钠。其处方符合药典要求，为每1g白蛋白添加0.159mmol辛酸钠，未添加N-乙酰色氨酸。pH为6.7，总钠量不超过160mmol/L。采用实施例1的方法测试样品3残留的长链脂肪的含量，长链脂肪酸比例(mol/mol人白蛋白)：0.34：1。
图3为样品3的动态光散色仪图谱。
按照实施例1中的方法去除样品3中的30nm-300nm颗粒，在处理后的人血清白蛋白中按照每克人白蛋白中添加100微克泊洛沙姆188(BASF批号WPAK527B)以得到样品4，其动态光散色仪图谱如图4所示。
图3和图4的动态光散色仪的结果表明，在25℃和60℃两个温度点上，未添加泊洛沙姆的市售人血清白蛋白在30nm-300nm之间出现微小颗粒。而除去颗粒后，少量添加泊洛沙姆的人血清白蛋白在25℃和60℃两个温度点上都未出现微小颗粒。
实施例1和2表明，药典中的两种标准人血清白蛋白处方，在不添加泊洛沙姆时都出现200nm左右的颗粒；添加少量泊洛沙姆后，都可以有效地抑制和阻止微小颗粒的出现。
本申请发明人通过试验还发现，作为该具体实施例的变形，泊洛沙姆188也可以替换成泊洛沙姆124、泊洛沙姆188、泊洛沙姆237、泊洛沙姆338或泊洛沙姆407中的一种，或者，也可以采用泊洛沙姆188、泊洛沙姆124、泊洛沙姆188、泊洛沙姆237、泊洛沙姆338或泊洛沙姆407中的多种同时使用。试验证明，这些泊洛沙姆也可以实现同样的效果，即除去颗粒后，少量添加泊洛沙姆的人血清白蛋白在25℃和60℃两个温度点上都未出现30nm-300nm之间的微小颗粒。
本申请发明人通过试验还发现，只需要添加少量的泊洛沙姆即可以达到理想的效果，通常，添加到10微克泊洛沙姆/克人白蛋白以上，基本上检测不到30nm-300nm之间的微小颗粒，或者，只出现很少的30nm-300nm之间的微小颗粒；添加量超过500微克泊洛沙姆/克人白蛋白时，30nm-300nm之间的微小颗粒会有明显的增多。
实施例3
按照申请号为201010124935.2的中国专利申请的方法，经改进纯化后得到的高纯度重组人白蛋白原液，经测试纯化后的重组人白蛋白长链脂肪酸残留量为长链脂肪酸比例(mol/mol人白蛋白)等于0.059：1，比人血清来源的长链脂肪酸含量低近10倍。未添加长链脂肪酸和泊洛沙姆，仅按照中国药典2015年版的方法要求制备样品5，为每1g白蛋白添加0.071mmol的辛酸钠(成都华邑药用辅料制造有限公司批号20170901)，每1g白蛋白添加0.085mmol的乙酰色氨酸(Adamas Reagent Co.,Ltd批号：P230264)，pH为6.8，控制总钠量不超过160mmol/L。
图5为未添加泊洛沙姆和长链脂肪酸的重组人白蛋白(样品5)的动态光散色仪图谱。
实施例4
样品6的制备：在上述实施例3方法得到的高纯度重组人白蛋白原液，在原液中添加油酸钠(BBI Life Sciences批号：E802BA0026)到长链脂肪酸/重组人白蛋白的摩尔比为1.0：1.0(原液残留长链脂肪酸计算在内)，每1g白蛋白添加0.081mmol的辛酸钠(成都华邑药用辅料制造有限公司批号20170901)，每1g白蛋白添加0.067mmol的乙酰色氨酸(Adamas Reagent Co.,Ltd批号：P230264)，pH为6.7，控制总钠量不超过160mmol/L。
图6为仅添加长链脂肪酸的重组含人白蛋白的制剂(样品6)的动态光散色仪图谱。
实施例5
样品7的制备：在上述实施例3方法得到的高纯度重组人白蛋白原液中，按照每克人白蛋白中添加100微克泊洛沙姆188(BASF批号WPAK527B)，每1g白蛋白添加0.079mmol的辛酸钠(成都华邑药用辅料制造有限公司批号20170901)，每1g白蛋白添加0.066mmol的乙酰色氨酸(Adamas Reagent Co.,Ltd批号：P230264)，pH为6.6，控制总钠量不超过160mmol/L。
图7为仅添加泊洛沙姆的重组人白蛋白(样品7)的动态光散色仪图谱。
实施例6
样品8的制备：在上述实施例3方法得到的高纯度重组人白蛋白原液中，按照每克人白蛋白中添加100微克泊洛沙姆188(BASF批号WPAK527B)，和添加油酸钠(BBI Life Sciences批号：E802BA0026)到长链脂肪酸/重组人白蛋白的摩尔比为1.0：1.0(原液残留长链脂肪酸计算在内)，以及每1g白蛋白添加0.083mmol的辛酸钠(成都华邑药用辅料制造有限公司批号20170901)，每1g白蛋白添加0.084mmol的乙酰色氨酸(Adamas Reagent Co.,Ltd批号：P230264)，pH为6.8，控制总钠量不超过160mmol/L。
图8为添加泊洛沙姆和长链脂肪酸的重组人白蛋白(样品8)的动态光散色仪图谱。
实施例7
样品9的制备：在上述实施例3方法得到的高纯度重组人白蛋白原液中，按照每克人白蛋白中添加100微克泊洛沙姆188(6BASF批号批号WPAK527B)，和添加大豆油酸钠(青岛瑞诺化工有限公司批号：20190121)到长链脂肪酸/重组人白蛋白的摩尔比为0.5：1.0(原液残留长链脂肪酸计算在内)，以及每1g白蛋白添加0.082mmol的辛酸钠(成都华邑药用辅料制造有限公司批号20170901)，每1g白蛋白添加0.080mmol的乙酰色氨酸(Adamas Reagent Co.,Ltd批号：P230264)， pH为6.9，控制总钠量不超过160mmol/L。
图9为添加泊洛沙姆和长链脂肪酸(大豆油酸钠)的重组人白蛋白(样品9)的动态光散色仪图谱。
实施例8
样品10的制备：在上述实施例3方法得到的高纯度重组人白蛋白原液中，按照每克人白蛋白中添加50微克泊洛沙姆188(BASF批号WPAK527B)，和添加按硬脂酸钠(Damas-beta批号：P1042477)和油酸钠(BBI Life Sciences批号：E802BA0026)1：2摩尔混合均匀后，按混合长链脂肪酸/重组人白蛋白的摩尔比为0.6：1.0(原液残留长链脂肪酸计算在内)，以及每1g白蛋白添加0.086mmol的辛酸钠(成都华邑药用辅料制造有限公司批号20170901)，每1g白蛋白添加0.076mmol的乙酰色氨酸(Adamas Reagent Co.,Ltd批号：P230264)，pH为7.0，控制总钠量不超过160mmol/L。
图10为添加泊洛沙姆和长链脂肪酸(硬脂酸钠/油酸钠混合)的重组人白蛋白(样品10)的动态光散色仪图谱。
实施例9
样品11的制备：在上述实施例3方法得到的高纯度重组人白蛋白原液中，按照每克人白蛋白中添加50微克泊洛沙姆188(BASF批号WPAK527B)，和添加按硬脂酸钠(Damas-beta批号：P1042477)和油酸钠(BBI Life Sciences批号：E802BA0026)1：2摩尔混合均匀后，按混合长链脂肪酸/重组人白蛋白的摩尔比为0.6：1.0(原液残留长链脂肪酸计算在内)，以及每1g白蛋白添加0.153mmol的辛酸钠(成都华邑药用辅料制造有限公司批号20170901)，pH为7.0，控制总钠量不超过160mmol/L。
图11为添加泊洛沙姆和添加长链脂肪酸(硬脂酸钠/油酸钠混合)的重组人白蛋白(样品11)的动态光散色仪图谱。
实施例10
实施例1-9制备的样品(样品1-11)的培育和热稳定试验。其方法参照《中国药典》2015年版三部第244页-第245页。其实验结果如表1所示。
表1样品1-11的稳定性观察结果
样品序列 57℃/50h培育 30℃/14天培育 20℃/28天培育
样品1 (-) (-) (-)
样品2 (-) (-) (-)
样品3 (-) (-) (-)
样品4 (-) (-) (-)
样品5 (+) (+) (+)
样品6 (+) (+) (+)
样品7 (+) (+) (+)
样品8 (-) (-) (-)
样品9 (-) (-) (-)
样品10 (-) (-) (-)
样品11 (-) (-) (-)
(注：-阴性，代表合格；+阳性，代表不合格。)
以上实施例证明了油酸、大豆油酸钠、硬脂酸钠/油酸钠混合、硬脂酸钠/油酸钠混合与泊洛沙姆联合使用对重组人白蛋白制剂的稳定性的促进作用。本申请发明人通过试验还发现，作为该具体实施例的变形，长链脂肪酸还可以采用肉豆蔻酸(C14:0)、棕榈酸(C16:0)、硬脂酸(C18:0)、油酸(C18:1)、亚油酸(C18:2)、亚麻酸(C18:3)、花生四烯酸酸(C20:4)中的一种或多种。试验证明，这些长链脂肪酸也可以实现同样的效果，即与泊洛沙姆联合使用促进重组人白蛋白制剂的稳定性。在上述稳定性测试中呈现阴性。
本申请发明人通过试验还发现，作为上述具体实施例的变形，泊洛沙姆188也可以替换成泊洛沙姆124、泊洛沙姆188、泊洛沙姆237、泊洛沙姆338或泊洛沙姆407中的一种，或者，也可以采用泊洛沙姆188、泊洛沙姆124、泊洛沙姆188、泊洛沙姆237、泊洛沙姆338或泊洛沙姆407中的多种同时使用。试验证明，这些泊洛沙姆也可以实现同样的效果，添加适量的长链脂肪酸后，少量添加泊洛沙姆的人血清白蛋白的稳定性可以达到要求。
本申请发明人通过试验还发现，只需要添加少量的泊洛沙姆即可以达到理想的效果，通常，添加到10微克泊洛沙姆/克人白蛋白以上，稳定性观察结果基本都为阴性；添加量超过500微克泊洛沙姆/克人白蛋白时，稳定性观察结果开始出现阳性。
本申请发明人通过试验还发现，长链脂肪酸与人白蛋白的摩尔比为0.2-3.0，尤其是为0.3-2.0时，人白蛋白制剂的稳定性处于最佳状态。当长链脂肪酸与人白蛋白的摩尔比低于0.3或高于2.0时，稳定性观察结果开始少量出现阳性；当长链脂肪酸与人白蛋白的摩尔比低于0.2或高于3.0时，稳定性观察结果开始大量出现阳性。
实施例11
将实施例3-9方法制备的样品5-11，进行差热扫描分析(DSC)图谱。DSC型号：Nano DSC 602000；厂家：TA；样品稀释液：0.01mol/L磷酸，pH中性。实验条件：将样品原液采用稀释液稀释至1-5mg/ml；升温范围：45℃-90℃；步长：1℃/min。其实验结果如表2所示。
表2样品5-11的差热扫描分析结果
样品序列 T onset T m1 T m2
高纯重组人白蛋白原液 60.9℃ 65.0℃ 70.1℃
样品5 62.9℃ 66.7℃ 70.5℃
样品6 63.2℃ 68.0℃ 73.7℃
样品7 62.8℃ 67.0℃ 71.6℃
样品8 63.3℃ 68.3℃ 74.2℃
样品9 64.0℃ 68.7℃ 72.9℃
样品10 63.6℃ 68.2℃ 70.0℃
样品11 64.7℃ 68.8℃ 73.1℃
本发明的上述结果表明，泊洛沙姆和长链脂肪酸有协同稳定性的效应。人血源白蛋白制品中自身残留的人体循环的长链脂肪酸和其他配体，可以不需要进一步添加长链脂肪酸，仅添加少量的泊洛沙姆就可以有效地抑制30nm-300nm颗粒的出现和提高热稳定性。
高度纯化的重组人白蛋白基本脱脂完全，需要添加泊洛沙姆抑制30nm-300nm颗粒的出现。对比实验结果证明：仅添加泊洛沙姆、常规药典的辛酸钠(中链脂肪酸)和乙酰色氨酸，中链脂肪酸尽管可以有效提高T onset、T m1和T m2，但是缺乏长链脂肪酸对白蛋白的各个相邻结构域配体相互支撑作用，依然不能满足热稳定性的要求。
因此，控制长链脂肪酸的含量能有效地提高T onset、T m1和T m2，增加蛋白质结构域的关联稳定性的同时需要添加少量的泊洛沙姆，以抑制疏水力的相互作用。本发明的含人白蛋白的制剂可以满足57℃/50h培育、30℃/14天培育和20℃/28天培育的稳定性要求，其稳定性指标优异，适合长期2℃-8℃、室温保存和运输的稳定性要求。
上述实施例仅例示性说明本发明的原理及其功效，而非用于限制本发明。任何熟悉此技术的人士皆可在不违背本发明的精神及范畴下，对上述实施例进行修饰或改变。 因此，举凡所属技术领域中具有通常知识者在未脱离本发明所揭示的精神与技术思想下所完成的一切等效修饰或改变，仍应由本发明的权利要求所涵盖。

Claims (35)

  1. 一种制备含人白蛋白的制剂的方法，其特征在于，所述方法包括：通过控制所述制剂中的长链脂肪酸的含量以及添加泊洛沙姆来提高所述制剂中的人白蛋白的稳定性。
  2. 如权利要求1所述的方法，其特征在于，所述方法还包括：通过减少人白蛋白的团聚和原纤维化来提高所述制剂中的人白蛋白的稳定性。
  3. 如权利要求1所述的方法，其特征在于，所述方法还包括：提高所述制剂中的人白蛋白的稳定性使得所述制剂制备完成后不出现由人白蛋白团聚和/或原纤维化形成的粒径在30nm以上的颗粒。
  4. 如权利要求1所述的方法，其特征在于，所述方法还包括：提高所述制剂中的人白蛋白的稳定性使得所述制剂制备完成后不出现由人白蛋白团聚和/或原纤维化形成的粒径在100nm以上的颗粒。
  5. 如权利要求1所述的方法，其特征在于，所述方法还包括：提高所述制剂中的人白蛋白的稳定性使得所述制剂制备完成后不出现由人白蛋白团聚和/或原纤维化形成的粒径在200nm左右的颗粒。
  6. 如权利要求1所述的方法，其特征在于，所述方法还包括：提高所述制剂中的人白蛋白的稳定性使得稳定性达到当地药典的要求。
  7. 如权利要求1所述的方法，其特征在于，所述方法还包括：提高所述制剂中的人白蛋白的稳定性使得稳定性达到如下条件：在57℃培育50h、30℃培育14天和20℃培育28天中不出现浑浊和可见异物。
  8. 如权利要求1所述的方法，其特征在于，所述人白蛋白为人血清白蛋白，所述方法包括：通过添加泊洛沙姆来提高所述制剂中的人白蛋白的稳定性。
  9. 如权利要求1所述的方法，其特征在于，所述人白蛋白为重组人白蛋白，所述方法包括：通过添加长链脂肪酸和泊洛沙姆来提高所述制剂中的人白蛋白的稳定性。
  10. 如权利要求9所述的方法，其特征在于，所述长链脂肪酸以盐的方式添加。
  11. 如权利要求9所述的方法，其特征在于，所述长链脂肪酸以钠盐的方式添加。
  12. 如权利要求1所述的方法，其特征在于，所述方法还包括：所述制剂制备完成后，所述长链脂肪酸与人白蛋白的摩尔比为0.2-3.0。
  13. 如权利要求1所述的方法，其特征在于，所述方法还包括：所述制剂制备完成后，所述长链脂肪酸与人白蛋白的摩尔比为0.3-2.0。
  14. 如权利要求1所述的方法，其特征在于，所述方法还包括：所述制剂制备完成后，所述泊洛沙姆的含量为10-500微克/克人白蛋白。
  15. 如权利要求1所述的方法，其特征在于，所述泊洛沙姆选自泊洛沙姆124、泊洛沙姆188、泊洛沙姆237、泊洛沙姆338和泊洛沙姆407中的一种或多种。
  16. 如权利要求1所述的方法，其特征在于，所述泊洛沙姆是泊洛沙姆188。
  17. 如权利要求1所述的方法，其特征在于，所述长链脂肪酸选自肉豆蔻酸(C14:0)、棕榈酸(C16:0)、硬脂酸(C18:0)、油酸(C18:1)、亚油酸(C18:2)、亚麻酸(C18:3)、花生四烯酸酸(C20:4)中的一种或多种。
  18. 如权利要求1所述的方法，其特征在于，所述长链脂肪酸为大豆油酸或全氢化大豆油酸。
  19. 如权利要求1所述的方法，其特征在于，所述方法还包括：添加当地药典所要求的成分以及含量。
  20. 如权利要求19所述的方法，其特征在于，当地药典所要求的成分包括辛酸钠或/和N-乙酰色氨酸。
  21. 根据如权利要求1所述的方法所制备的含人白蛋白的制剂。
  22. 一种含人白蛋白的制剂，其特征在于，所述制剂不含有由人白蛋白团聚和/或原纤维化形成的粒径在30nm以上的颗粒。
  23. 一种含人白蛋白的制剂，其特征在于，所述制剂不含有由人白蛋白团聚和/或原纤维化形成的粒径在100nm以上的颗粒。
  24. 一种含人白蛋白的制剂，其特征在于，所述制剂不含有由人白蛋白团聚和/或原纤维化形成的粒径在200nm左右的颗粒。
  25. 一种含人白蛋白的制剂，其特征在于，所述方法还包括：所述制剂中的人白蛋白的稳定性达到如下条件：在57℃培育50h、30℃培育14天和20℃培育28天中不出现浑浊和可见异物。
  26. 一种含人白蛋白的制剂，其特征在于，所述制剂中含有泊洛沙姆和长链脂肪酸。
  27. 如权利要求26所述的制剂，其特征在于，所述制剂中的长链脂肪酸与人白蛋白的摩尔比为0.2-3.0。
  28. 如权利要求26所述的制剂，其特征在于，所述制剂中的长链脂肪酸与人白蛋白的摩尔比为0.3-2.0。
  29. 如权利要求26所述的制剂，其特征在于，所述方法还包括：所述制剂中的泊洛沙姆的含量为10-500微克/克人白蛋白。
  30. 如权利要求26所述的制剂，其特征在于，所述泊洛沙姆选自泊洛沙姆124、泊洛沙姆188、泊洛沙姆237、泊洛沙姆338和泊洛沙姆407中的一种或多种。
  31. 如权利要求26所述的制剂，其特征在于，所述泊洛沙姆是泊洛沙姆188。
  32. 如权利要求26所述的制剂，其特征在于，所述长链脂肪酸选自肉豆蔻酸(C14:0)、棕榈酸(C16:0)、硬脂酸(C18:0)、油酸(C18:1)、亚油酸(C18:2)、亚麻酸(C18:3)、花生四烯酸酸(C20:4)中的一种或多种。
  33. 如权利要求26所述的制剂，其特征在于，所述长链脂肪酸为大豆油酸或全氢化大豆油酸。
  34. 如权利要求26所述的制剂，其特征在于，所述制剂还含有当地药典所要求的成分以及含量。
  35. 如权利要求34所述的制剂，其特征在于，当地药典所要求的成分包括辛酸钠或/和N-乙酰色氨酸。
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