WO2014098464A1 - Human serum albumin heat-stabilizing composition, and production method for heat-stabilized human serum albumin using same - Google Patents

Abstract

The present invention relates to: a heat-stabilizing composition for producing a human serum albumin preparation, the composition comprising a fatty acid selected from the group consisting of C16 to C22 fatty acids, or a pharmaceutically acceptable salt thereof; a production method for heat-stabilized human serum albumin, the method comprising a first step of adjusting the human serum albumin concentration to between 1 and 40 mg/ml, and a second step of adding an above fatty acid or pharmaceutically acceptable salt thereof to the human serum albumin; and a production method for a human serum albumin preparation, the method comprising a heat-treatment step in which an above fatty acid or pharmaceutically acceptable salt thereof is treated in human serum albumin.

Description

Composition for heat stabilization of human serum albumin and method for preparing thermostable albumin for thermostabilization using same

The present invention comprises a composition for thermal stabilization for preparing a human serum albumin preparation comprising a fatty acid selected from the group consisting of fatty acids having 16 to 22 carbon atoms or a pharmaceutically acceptable salt thereof; A first step of adjusting the concentration of human serum albumin to 1 to 40 mg / ml; And a second step of adding the fatty acid or a pharmaceutically acceptable salt thereof to human serum albumin; And treating the human serum albumin with the fatty acid or a pharmaceutically acceptable salt thereof to heat-treat the human serum albumin.

Albumin is a protein found in the blood and is produced by the liver. Structurally, human serum albumin (HSA) consists of 585 amino acids (66,438 Da) and consists of 17 disulfide bridges and one free cysteine (Cys34) [Dugiaczyk, A. et. al. , Proc. Natl Acad. Sci. USA, 1982, 79: 71-75. In addition, albumin plays a role in maintaining and restoring plasma volume, in combination with various ligands such as cations such as water, calcium, sodium, potassium, fatty acids, hormones, bilirubin, and drugs to regulate and deliver colloidal osmotic pressure in the blood, and Malnutrition serves as a source of amino acids [Bar-Or, D. et al. , Eur J. Biochem., 2001, 268: 42-47]. In particular, the combination of the drug and albumin is greatly involved in the drug expression of the drug. Purified HSAs, on the other hand, are used for the treatment of hypoalbuminemia due to albumin loss and albumin synthetic dysfunction, such as, for example, surgery, hemorrhagic shock or burns and nephrotic syndromes. Albumin is also used as a pharmaceutical additive in formulating protein for supplementation and treatment of media used for the growth of higher eukaryotic cells. Another known function of serum albumin is that it acts as an oxygen carrier that adsorbs and desorbs oxygen under oxygen partial pressure, similar to hemoglobin. It can therefore be administered in place of hemoglobin in an emergency. Currently, the demand for albumin products is met by albumin extracted from human blood.

Conventional HSA is a low-temperature ethanol fraction of Con (Cone) from human plasma or a method equivalent thereto, after obtaining an HSA-containing fraction (fraction V), it is prepared through various purification methods. As a purification method, purification methods generally used in protein chemistry, for example, salting out method, ultrafiltration, isoelectric point precipitation, electrophoresis, ion exchange chromatography, gel filtration chromatography, Affinity chromatography and the like can be used. In fact, since many kinds of contaminants derived from biological tissues, cells, blood, and the like are mixed in a sample obtained from plasma, it is necessary to use human serum albumin in combination. However, the amount of albumin purified from human plasma is inevitably limited. Therefore, in recent years, yeast [Ken, O. et al. , J. Biochem., 1991, 110: 103-110] and Bacillus subtilis [Saunders, CW et al. , J. Bacteriol., 1987, 169: 2917-2925, have been developed for the production of human serum albumin.

Typically, albumin preparations in aqueous form are subjected to a heat treatment at 60 ° C. for 10 hours to inactivate viruses that may contaminate the preparation. In addition, industrial production of albumin is carried out under conditions different from the environment in the human body. Therefore, considering the properties of albumin which are easily denatured and aggregated by heat or the like, there is a high possibility of denaturation or multimer formation during this series of processes. Currently, commercially available 5% or 20% albumin preparations are widely used without any side effects, and the multimers contained in the preparations are considered to be harmless to humans.However, blood pressure rise, fever, tachycardia, tremor, flushing and hives caused by denatured albumin injections , Side effects such as chills have been reported. Thus, albumin preparations currently on the market to prevent denaturation by heat treatment are prepared by adding sodium octanoate and N-acetyl tryptophan as stabilizers.

Given the continued research on the various net functions of albumin and the possibility of developing new albumin preparations, the demand is expected to continue to increase. However, at present, the only source of human serum albumin is human blood, which is rather limited, and therefore it is urgent to develop new sources as well as to study the properties of albumin. Given the current high technology in life science and technology, recombinant human serum albumin using transformants may be another source for the production of albumin preparations.

On the other hand, as described above albumin exhibits physiological activity in combination with various substances such as water, ions, fatty acids, hormones. Therefore, in order to utilize recombinant human serum albumin in the production of albumin preparations, it is necessary to identify the elements necessary for albumin to exhibit its physiological activity and to identify how they bind, and apply it to recombinant human serum albumin to have physiological activity. It is necessary.

The present inventors have made efforts to identify ligands that naturally bind to human serum albumin, which is naturally physiologically active in vivo, and to find a substance capable of stabilizing albumin against heat treatment, which is an essential process of the albumin preparation process. , Human serum albumin is combined with saturated or unsaturated fatty acids having 16 to 22 carbon atoms such as oleic acid and linoleic acid, and the fatty acid improves the thermal stability to albumin, thereby preventing denaturation during the heat treatment process required for the production of albumin preparations It was confirmed that can be represented. Furthermore, when the fatty acid or its salt is added to human serum albumin at a concentration of 1 to 40 mg / ml, it was confirmed that stability can be maintained high even after a long heat treatment for about 10 hours, thereby completing the present invention.

One object of the present invention is to provide a composition for thermal stabilization for preparing a human serum albumin preparation comprising a fatty acid selected from the group consisting of fatty acids having 16 to 22 carbon atoms or a pharmaceutically acceptable salt thereof.

Another object of the present invention is the first step of adjusting the concentration of human serum albumin to 1 to 40 mg / ㎖; And a second step of adding a fatty acid selected from the group consisting of fatty acids having 16 to 22 carbon atoms or a pharmaceutically acceptable salt thereof to human serum albumin, the method of preparing heat stabilized human serum albumin.

Another object of the present invention is a method for preparing a human serum albumin preparation, the step of treating the human serum albumin by heat treatment with a fatty acid selected from the group consisting of fatty acids having 16 to 22 or a pharmaceutically acceptable salt thereof It provides a method for producing a human serum albumin containing.

Adding a fatty acid selected from the group consisting of fatty acids having 16 to 22 carbon atoms according to the present invention, it can be usefully used to produce stable albumin even during the heat treatment process in the preparation of human serum albumin preparation. In particular, by adding the fatty acid to human serum albumin at a concentration of 1 to 40 mg / ml, even when heat-treated at a high temperature of 60 ° C. or higher for 10 hours or more, human serum albumin with stable stability can be prepared. On the other hand, when recombinant human serum albumin is used, it is possible to produce recombinant human serum albumin that is most structurally similar to that of human origin, so it can be used industrially without side effects, and even when using plasma-derived human serum albumin, it is not a foreign substance. By using a composition containing various fatty acids naturally present in the preparation, the preparation can be made without worrying about toxicity or side effects.

1 is a diagram showing a three-dimensional conformation of human serum albumin crystals obtained from a 20% human serum albumin injection of Green Cross, which is prepared from plasma-derived human serum albumin, which is obtained by X-ray crystallography. . The stick position of L-tryptophan, which is a decomposition product of octanoic acid and N-acetyl tryptophan, used as a heat stabilizer in the manufacturing process of plasma-derived human serum albumin, Indicated. The binding position of oleic acid, which is one of the C18 fatty acids, is represented by a sphere model.

Figure 2 is a diagram showing the mass spectrometry results of the 20% human serum albumin injection of Green Cross, which is formulated and commercialized using plasma-derived human serum albumin.

FIG. 3 is a diagram showing the mass spectrometry results of a 20% human serum albumin injection prepared by commercially available plasma-derived human serum albumin.

4 is a view showing the sustainable treatment time according to the temperature of the fatty acid free human serum albumin solution and the human serum albumin solution added with oleic acid in minutes and log units thereof.

FIG. 5 shows five human serum albumin samples (Green Cross Albumin, A3782 Albumin, A1653 Albumin, Green Cross Albumin + Oleic Acid (1: 1 molar ratio), A3782 Albumin + Oleic Acid (1: 1 molar ratio) when heated at 60 ° C. for 10 hours. The figure which shows the result of having measured circular dichroism.

Figure 6 shows five human serum albumin samples (green cross albumin, A3782 albumin, A1653 albumin, green cross albumin + oleic acid (1: 1 molar ratio), A3782 albumin + oleic acid (1: 1 molar ratio) when heated at 70 ℃ for 10 hours The figure which shows the result of having measured circular dichroism.

FIG. 7 shows five human serum albumin samples (Green Cross Albumin, A3782 Albumin, A1653 Albumin, Green Cross Albumin + Oleic Acid (1: 1 molar ratio), A3782 Albumin + Oleic Acid (1: 1 molar ratio) when heated at 80 ° C. for 10 hours. The figure which shows the result of having measured circular dichroism.

FIG. 8 shows the results of measurement of circular dichroism when 10 human serum albumin samples having different concentrations (1 to 45 mg / ml A3782 albumin + oleic acid (1: 1 molar ratio)) were heated at 70 ° C. for 10 hours. It is also.

Figure 9 shows five human serum albumin samples with different fatty acids (A3782 albumin + oleic acid (1: 1 molar ratio), A3782 albumin + linoleic acid (1: 1 molar ratio), A3782 albumin + α-linolenic acid (1: 1 molar ratio), A3782 The results of measurement of circular dichroism when albumin + γ-linolenic acid (1: 1 molar ratio) and A3782 albumin + palmitoleic acid (1: 1 molar ratio) were heated at 70 ° C for 10 hours.

As one embodiment for achieving the above object, the present invention comprises a fatty acid selected from the group consisting of fatty acids having 16 to 22 carbon atoms or a pharmaceutically acceptable salt thereof, for thermal stabilization for preparing a human serum albumin preparation To provide a composition.

The use of recombinant human serum albumin in the method for producing human serum albumin preparation of the present invention can solve the problem of limited source in the preparation of conventional plasma-derived albumin preparation. However, since human serum albumin binds to various ligands in vivo to form a stable structure and exhibits physiological activity, in order to formulate recombinant human serum albumin, the structure of the bioactive albumin and / or the ligand bound thereto must be preceded. something to do. Meanwhile, in the conventional method for preparing a preparation using plasma-derived human serum albumin, foreign substances such as sodium octanoate and N-acetyl tryptophan, which have not been tested for stability, have been added to impart stability to heat treatment. There is a need to discover substances that exist naturally in living organisms. Accordingly, the present inventors have confirmed that recombination by confirming that fatty acids selected from the group consisting of fatty acids having 16 to 22 carbon atoms are bound to plasma-derived human serum albumin, and that they can impart stabilization of human serum albumin, in particular, stability to heat treatment. The addition of a fatty acid selected from the group consisting of fatty acids having 16 to 22 carbon atoms to human serum albumin can form stable albumin even during the heat treatment process, it was confirmed that the preparation of the preparation using the same.

The term "albumin" of the present invention is one of the proteins constituting the basic substance of the cell, is present in the blood very much, it is produced in the liver. It has the lowest molecular weight of simple proteins in nature. Serum albumin in the blood maintains and restores the plasma volume, preventing shock from excessive bleeding, and is used for surgery and burn treatment. It is also known to have oxygen transfer capacity similar to hemoglobin. For the purpose of the present invention, the composition of the present invention is to impart stability to heat treatment in the process of formulating the albumin, the albumin of the subject may include all the albumin that can be formulated, preferably human plasma Or recombinant human serum albumin produced by genetic engineering.

As used herein, the term "recombinant human serum albumin" is a term used as a concept for contrasting with natural human serum albumin derived from human plasma, and refers to human serum albumin produced artificially. For example, human serum albumin obtained from a transformant prepared to express a polypeptide having the amino acid sequence of SEQ ID NO: 1 (HSA sequence) may have the same amino acid sequence as human serum albumin isolated from plasma, but is not limited thereto. It doesn't work.

The term "transformant" of the present invention is an organism that has been genetically modified to express a particular gene through genetic engineering, for the purposes of the present invention, preferably the particular gene is It may be a gene encoding an amino acid sequence of SEQ ID NO: 1, such as human serum albumin. Preferably, the genetically modified organism is transformed into bacteria, fungi and yeasts. It may be a cell or a transgenic animal, but is not limited thereto. Preferably, as the transgenic animal, mammals such as mice, rats, and guinea pigs may be used, but are not limited thereto.

"Human serum albumin formulations" of the present invention is a concept including all formulations made with human serum albumin, which may be formulated in a form that can be administered to an individual including a human, and may include albumin formulations currently on the market. It may also include formulations having similar ingredients, functions, and medicaments as commercially available albumin formulations, prepared to replace them. Commercially available albumin preparations have a problem of relying on a limited source of blood, and as an alternative to solve this, recombinant human serum albumin preparations using recombinant human serum albumin may be prepared. Preferably, the recombinant human serum albumin preparation of the present invention may include recombinant human serum albumin obtained from a transformant prepared to express human serum albumin of SEQ ID NO: 1 as an active ingredient.

In order to prepare the human serum albumin as an agent that can be administered to the human body, a heat treatment process is required to prevent contamination of the virus and / or other infections. However, similar to normal proteins, human serum albumin may cause degeneration such as structure change or aggregation when treated at high temperatures. Therefore, an additive for stabilizing this is required, and octanates such as sodium octanoate and N-acetyl tryptophan are used in the preparation process of plasma-derived albumin. However, these substances are not added to the original plasma, but are added arbitrarily to improve the stability of the heat treatment process. Unlike natural human serum albumin, these substances may cause side effects when administered into the human body. There is this.

Thus, where possible, there is a need to maintain the form and structure most similar to natural human serum albumin in the preparation of human plasma-derived or recombinant human serum albumin preparations. In addition, since recombinant human serum albumin has some properties different from human serum albumin extracted from human plasma, it is more preferable to treat a substance present in the living body rather than any additives as a composition for thermostabilization to prepare a recombinant human serum albumin preparation. It can be safe. In the present invention, it was confirmed that the binding to the human serum albumin separated from the plasma fatty acids having 16 to 22 carbon atoms identified.

As used herein, the term "fatty acid having 16 to 22 carbon atoms" refers to a fatty acid having 16 to 22 carbons among fatty acids, and includes one end with a hydrophilic carboxyl group, and the other end is composed of a hydrophobic carbon chain. At this time, the type can be varied depending on the number and position of the double bonds in the carbon chain, preferably palmitic acid (palmitic acid) and palmitoleic acid (palmitoleic acid), a fatty acid of 16 carbon atoms, stearic acid of 18 fatty acids (stearic acid; octadecanoic acid), oleic acid, linoleic acid and linolenic acid, eicosapentaenoic acid (EPA), a fatty acid with 20 carbon atoms, and docosa, a fatty acid with 22 carbon atoms It may be selected from hexaenoic acid (DHA; docosahexaenoic acid), or a combination thereof, but is not limited thereto. The fatty acids may also be used in the form of pharmaceutically acceptable salts.

In the present invention, the term "palmitic acid" is a saturated fatty acid having 16 carbon chains and is called hexadecanoic acid in the name of IUPAC. It has the formula CH ₃ (CH ₂ ) ₁₄ CO ₂ H and is the most common fatty acid found in flora and fauna and microorganisms. It is also present in palm oil, meat, cheese, butter and dairy products. The salt and ester forms of palmitic acid are called palmitates. At basic pH it is present in the form of palmitate anion. Excess carbohydrates are converted to palmitic acid in the body. Palmitic acid is the first fatty acid produced during fatty acid synthesis and is a precursor to long-chain fatty acids. Biologically, some proteins can be modified by binding to palmitoyl groups and this is called palmitoylation, which is important for protein localization of proteins.

As used herein, the term "palmitoleic acid" is also referred to as omega-7 monounsaturated fatty acid, cis-palmitoleic acid, and 9-cis-hexadecenoic acid. The IUPAC name is hexadec-9-enoic acid. A fatty acid of carbon 16 including one double bond has a chemical formula of CH ₃ (CH ₂ ) ₅ CH = CH (CH ₂ ) ₇ CO ₂ H and is a component of glyceride of human adipose tissue. It is present in all tissues and is found in high concentrations in the liver. It is biosynthesized from palmitic acid by the action of delta-9 desaturase. Beneficial fatty acids not only inhibit the destruction of insulin-secreting isabeta cells, but also inhibit inflammation and increase insulin sensitivity. Edible palmitoleic acid can be extracted from the oils of plants and animals.

In the present invention, the term "stearic acid" is a saturated fatty acid having 18 carbon chains and is referred to as octadecanoic acid in the name of IUPAC. It is a waxy solid with the formula CH ₃ (CH ₂ ) ₁₆ CO ₂ H. Its salts and esters are called stearate. The stearic acid, together with palmitic acid, is one of the most widely saturated fatty acids in nature. It has dual functionality because it has a polar head that can bind metal ions and a nonpolar chain that provides solubility in organic solvents. Due to these properties, they can be used as surfactants or softeners.

As used herein, the term "oleic acid" is a colorless odorless oil produced from fats and oils of various plants and animals. Commercially available products may be yellow in color. It is chemically classified as monounsaturated omega-9 fatty acid and has the formula CH ₃ (CH ₂ ) ₇ CH = CH (CH ₂ ) ₇ CO ₂ H. Oleic acid, meanwhile, is the most abundant fatty acid in human adipose tissue.

As used herein, the term "linoleic acid" is an unsaturated n-6 fatty acid and a colorless liquid at room temperature. Linoleic acid is one of the essential fatty acids that cannot be synthesized from other food components in the body. In particular, it is a polyunsaturated fatty acid used for the biosynthesis of arachidonic acid and some prostaglandins. Found in cell membrane lipids and abundant in vegetable oils. Chemically a carboxylic acid with a chain of 18 carbons and two cis double bonds, the first double bond is located at the sixth carbon from the hydrophobic methyl end, CH ₃ (CH ₂ ) ₄ CH = CHCH ₂ CH = CH (CH ₂ ) ₇ COOH.

As used herein, the term "linolenic acid" refers to two forms of linolenic acid, α and γ, or a mixture thereof. The linolenic acid is usually present in the vegetable oil in the form of linolenate. Double α-linolenic acid is a carboxylic acid with 18 carbon chains and 3 cis double bonds, the first double bond being a polyunsaturated n-3 fatty acid located at the methyl end, i. Has the chemical name of all-cis-9,12,15-octadecatrienoic acid. Its isomer, γ-linolenic acid, is a polyunsaturated n-6 fatty acid having the same carbon number chain containing the same number of double bonds as the α-linolenic acid, but the first double bond being located at the 6th carbon from the n-terminal. The positions of the double bonds in the chains are different. γ-linolenic acid, also called gamolenic acid, is known to exhibit effects on inflammatory and autoimmune diseases.

In the present invention, the term "EPA (eicosapentaenoic acid)" is an omega-3 fatty acid, also called timnodonic acid, and the chemical structure is a carboxylic acid having five cis double bonds on 20 carbon chains. . The first double bond is located at the omega end, the third carbon from the hydrocarbon end. EPA is a polyunsaturated fatty acid (PUFA), which is the eicosanoid prostaglandin-3 (platelet coagulation inhibition), thromboxane-3 and leukotriene-5 Acts as a precursor. It is present in large amounts in seaweeds and fish, especially fish oils, and also in human breast milk. Fish do not naturally produce EPA, but are obtained from algae they consume. It is possible to convert alpha-linolenic acid to EPA in humans, but the efficiency is very low. EPA, on the other hand, acts as a precursor of DHA. The EPA has the formula CH ₃ CH ₂ (CH = CHCH ₂ ) ₅ (CH ₂ ) ₂ COOH.

As used herein, the term "DHA" (docosahexaenoic acid) is an omega-3 fatty acid that is also a primary component of the human brain, cerebral cortex, skin, sperm, testis and retina. It can be synthesized from alpha-linolenic acid or obtained directly from breast milk or fish oil. The chemical structure is a carboxylic acid with six cis double bonds on 22 carbon chains. The first double bond is located at the omega end, the third carbon from the hydrocarbon end. Common names are cervonic acid, IUPAC names (4Z, 7Z, 10Z, 13Z, 16Z, 19Z) -docosa-4,7,10,13,16,19-hexaenoic acid ((4Z, 7Z, 10Z, 13Z, 16Z, 19Z) -docosa-4,7,10,13,16,19-hexaenoic acid). It is known to be abundant in deep sea fish oil. It is derived from photosynthetic and other nutrient microalgae and concentrated in organisms located at the top of the food chain. It can be produced by the conversion of alpha-linolenic acid in the body, which conversion is about 15% higher in women and the conversion to DHA is reduced when testosterone is administered or the conversion of testosterone to estradiol is reduced. As the main fatty acids in sperm, brainphospholipids and retinas, ingesting DHA can reduce the risk of heart disease by reducing blood triglyceride levels. In addition, sub-normal DHA levels are also associated with Alzheimer's.

The fatty acids may be represented by N: X (n-Y) by the notation according to the number of lipids. N represents the carbon number of the entire chain, that is, 16, 18, 20 or 22, X represents the number of double bonds contained in the chain, Y represents the position of the carbon from which the double bond starts from the methyl terminal of each fatty acid. Ω may be written in the n dash. For example, since palmitic acid of the present invention is a saturated fatty acid having 16 carbon atoms, 16: 0, and palmitoleic acid have one double bond to the seventh carbon from the methyl terminal, so that 16: 1 (n-7) or 16: 1 (ω- 7) Since stearic acid is a saturated fatty acid of 18 carbon atoms, 18: 0, oleic acid has one double bond from the methyl terminal to the ninth carbon, so 18: 1 (n-9) or 18: 1 (ω-9), linoleic acid Is 18: 2 (n-6) or 18: 2 (ω-6), linolenic acid-α is 18: 3 (n-3) or 18: 3 (ω-3), and linolenic acid-γ is 18: 3 (n -6) or 18: 3 (ω-6), EPA is 20: 5 (n-3) and finally DHA is 22: 6 (n-3).

According to one embodiment of the present invention, as a result of mass spectrometry and mass spectrometry analysis of the injection-derived plasma-derived human serum albumin, it was confirmed that 18 fatty acids including oleic acid are bound (FIGS. 2 and 3). ). In addition, the binding position of the fatty acid to albumin was confirmed (FIG. 1). Thus, oleic acid or other fatty acid having 18 carbon atoms, fatty acid having 16 carbon atoms, EPA or DHA is added to a fatty acid free human serum albumin solution and subjected to high temperature treatment, and human serum albumin added with oleic acid having 18 carbon atoms is oleic acid. Compared to the free fatty acid free human serum albumin solution, it was confirmed that even at a high temperature of 70 ° C., it could maintain a stable form without degeneration for a long time (more than 2 times) (Tables 3 to 6). It was confirmed that the added albumin solution can be maintained without deformation for a longer time compared to the fatty acid free albumin solution (Table 7). In addition, it was confirmed that the same effect is shown for fatty acids having 16 carbon atoms, EPA and DHA (Tables 8 and 9).

The term "pharmaceutically acceptable salts" of the present invention retains the desired biological and / or physiological activity of the composition and minimizes the unwanted toxicological effects, which are not toxic to cells or humans exposed to the composition. Means all salts represented. As salts are acid addition salts formed with pharmaceutically acceptable free acids. Acid addition salts are prepared by conventional methods, for example by dissolving a compound in an excess of aqueous acid solution and precipitating the salt using a water miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. Equivalent molar amounts of the compound and acid or alcohol (eg, glycol monomethyl ether) in water can be heated and the mixture can then be evaporated to dryness or the precipitated salts can be suction filtered. In this case, inorganic acids and organic acids may be used as the free acid, and hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, tartaric acid, and the like may be used as the inorganic acid, and methane sulfonic acid, p-toluene sulfonic acid, acetic acid, and triacid may be used as the organic acid. Fruroacetic acid, maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, manderic acid, propionic acid, citric acid, lactic acid, glycolic acid ( glycollic acid, gluconic acid, galacturonic acid, glutamic acid, glutaric acid, glucuronic acid, aspartic acid, ascorbic acid, carbonic acid, vanillic acid, hydroiodic acid, etc. Can be used, but not limited to these.

In addition, bases may be preferably used to make pharmaceutically acceptable metal salts. Alkali metal or alkaline earth metal salts are obtained, for example, by dissolving a compound in an excess of alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the insoluble compound salt, and then evaporating and drying the filtrate. In this case, as the metal salt, it is particularly suitable to prepare sodium, potassium or calcium salt, but is not limited thereto. Corresponding silver salts may also be obtained by reacting alkali or alkaline earth metal salts with a suitable silver salt (eg, silver nitrate).

The fatty acids having 16 to 22 carbon atoms or pharmaceutically acceptable salts thereof include salts of acidic or basic groups which may be present in the fatty acids having 16 to 22 carbon atoms unless otherwise indicated. For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts of the hydroxy group, and other pharmaceutically acceptable salts of the amino group include hydrobromide, sulfate, hydrogen sulphate, phosphate, hydrogen phosphate, Hydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate (mesylate) and p-toluenesulfonate (tosylate) salts; and the like through the methods for preparing salts known in the art. Can be prepared. Preferably, it may be an alkali metal or alkaline earth metal salt, but is not limited thereto.

Preferably, the composition for thermal stabilization of the present invention may be added such that the fatty acid or pharmaceutically acceptable salt thereof is in a molar ratio of 1: 0.5 to 1: 2 with respect to albumin, and preferably 1: 0.7 to 1: 1.5, more preferably 1: 0.8 to 1: 1.2, even more preferably may be added to a molar ratio of 1: 1, but is not limited thereto.

In another embodiment, the present invention comprises the steps of adjusting the concentration of human serum albumin to 1 to 40 mg / ㎖; And a second step of adding a fatty acid selected from the group consisting of fatty acids having 16 to 22 carbon atoms or a pharmaceutically acceptable salt thereof to human serum albumin, the method of preparing heat stabilized human serum albumin.

The fatty acids having 16 to 22 carbon atoms, or pharmaceutically acceptable salts thereof, preferred amounts thereof, and human serum albumin are as described above.

Previously reported side effects of infectious diseases of albumin, as a way to overcome this, introduced a heat treatment process at 60 ℃ with a virus killing effect by cold ethanol for 10 hours. However, although albumin is a structurally very stable protein, the formation of dimers, oligomers and polymers may be induced during the heat treatment, and in severe cases, gelation has been reported. Therefore, in the preparation of human serum albumin preparations, it is important to develop a method capable of maintaining the stability of human serum albumin for a long time in the required heat treatment step. Therefore, in the present invention, when treated with a low concentration of human serum albumin of 1 to 40 mg / ㎖ of 16 to 22 carbon atoms or a pharmaceutically acceptable salt thereof, even if the heat treatment for 10 hours at a temperature of 60 to 80 ℃ The secondary structure of albumin was maintained. Such a method of the present invention may have a usefulness for the development of albumin preparations having increased stability by imparting stability of the albumin structure to the heat treatment process for several hours.

Preferably, the first and second steps may be performed in sequence or in reverse order. In the method for preparing thermostable human serum albumin of the present invention, the concentration of human serum albumin sample containing 16 to 22 carbon atoms is 1-40 mg / ml. When the heat treatment after adjusting to a low level, it is characterized in that the stability can be maintained even after several hours of heat treatment. In this case, the fatty acid having 16 to 22 carbon atoms to be added is 1: 0.5 to 1: 2, preferably 1: 0.7 to 1: 1.5, more preferably 1: 0.8 to 1: 1.2, and even more preferably human serum albumin. Preferably a molar ratio of 1: 1. Therefore, the second step of adding fatty acids having 16 to 22 carbon atoms to human serum albumin is before or after adjusting the concentration of human serum albumin regardless of the order, as long as the final concentration of human serum albumin is maintained at the above level. Can be performed.

Α-helical structure of human serum albumin prior to heating up to 5 to 16 hours when heat is applied to low-concentration human serum albumin of 1 to 40 mg / ml of the C16-C24 fatty acid according to the preparation method of the present invention. up to 80 to 100% of (α-Helix).

At this time, in order to adjust the concentration of the human serum albumin to 1 to 40 mg / ㎖, various dilution buffers generally used in the preparation of albumin preparations can be used. Examples of such dilution buffers may include histidine-buffer, citrate-buffer, succinate-buffer, acetate-buffer or phosphate-buffer, and the like, preferably 50 mM potassium phosphate and 150 mM NaCl (pH 7.5). It may be a mixed solution of, but is not limited thereto.

Since heat-stabilized human serum albumin provided according to the method of the present invention is intended to be formulated as described above and administered to humans, the preparation method thereof may include a heat treatment process to block the possibility of contamination and / or infection. .

Therefore, in order to formulate it may include the step of heating the human serum albumin to 60 to 80 ℃, preferably may be heated to 65 to 75 ℃, more preferably 70 ℃. At this time, the thermostable human serum albumin according to the present invention, even when heated at the temperature for 6 hours to 15 hours, preferably 8 hours to 12 hours, more preferably 10 hours, α of the human serum albumin It is characterized by the fact that the spiral structure (α-Helix) can be maintained.

In one embodiment of the present invention, after treatment with human serum albumin at a concentration of 1 mg / ㎖ oleic acid in a molar ratio of 1: 1 and heated at 60 to 80 ℃ for 10 hours, α-helix from the measurement of circular dichroism As a result of observing the structural stability of albumin through the composition ratio of the structure, it was confirmed that the stability was maintained at 60 ℃ and 70 ℃, but the stability was reduced above 80 ℃ (Example 7, Figures 5 to 7).

In addition, it was confirmed that even after treatment with oleic acid in a molar ratio of 1: 1 to human serum albumin at a concentration of 1 to 40 mg / ml and heating at 70 ° C. for 10 hours (Example 8, FIG. 8), oleic acid In addition, after treating the fatty acids of linoleic acid, α-linolenic acid, γ-linolenic acid and palmitoleic acid typically, and after heating for 10 hours at 70 ℃, it was confirmed that the stability is maintained (Example 9, Figure 9).

 This suggests that the thermal stability of low concentrations of human serum albumin of 1 to 40 mg / ml is significantly increased by the addition of 16 to 24 carbon atoms such as oleic acid.

In still another aspect, the present invention provides a method for preparing a human serum albumin preparation, wherein the human serum albumin is heat treated by treating a fatty acid selected from the group consisting of fatty acids having 16 to 22 carbon atoms or a pharmaceutically acceptable salt thereof. It provides a method for producing a human serum albumin preparation comprising the step.

The fatty acids having 16 to 22 carbon atoms, or pharmaceutically acceptable salts thereof, preferred amounts thereof, and human serum albumin are as described above.

As used herein, the term "treatment" is a general term for all processes of including a fatty acid having 16 to 24 carbon atoms or a pharmaceutically acceptable salt thereof in any form, such as chemical or biological action, for thermal stabilization. For example, it may be applied at any stage prior to heat treatment, such as incorporating it into the medium composition during the culturing to prepare heat stabilized human serum albumin, or in the form of the composition in the processing of human serum albumin.

Preferably, the human serum albumin preparation according to the present invention may be prepared using albumin isolated from human plasma or recombinant human serum albumin prepared by genetic engineering.

Since the human serum albumin preparation according to the present invention aims to be administered to humans as described above, the preparation method thereof includes a heat treatment process for blocking the possibility of contamination and / or infection. Accordingly, in order to prevent denaturation of the protein by the heat treatment process, the step of thermally stabilizing by treating the human serum albumin fatty acid selected from the group consisting of fatty acids having 16 to 22 or a pharmaceutically acceptable salt thereof Is characteristic.

In the conventional manufacturing process of human serum albumin, sodium octanoate and / or N-acetyl tryptophan are used to impart stability to heat treatment, but in the present invention, C16-C22 fatty acids have the same or superior heat compared to the compounds. It has been confirmed that it has a stabilizing effect, and in addition, the fatty acid having 18 carbon atoms has the advantage of being able to maintain the most similar form and structure to natural human serum albumin without any side effects included in the human serum albumin preparation.

In preparing a preparation of the present invention, when using recombinant human serum albumin, the recombinant human serum albumin may be obtained from a transformant expressing human serum albumin, which is preferably a polypeptide having an amino acid sequence of SEQ ID NO: 1. have. The definition of the transformant is the same as described above.

As used herein, the term "treatment" is a general term for all processes of including a fatty acid having 16 to 22 carbon atoms or a pharmaceutically acceptable salt thereof in any form, such as chemical or biological action, for thermal stabilization. For example, it may be applied at any stage before heat treatment, such as inclusion in the medium composition in the culture process for producing human serum albumin, or in the form of the composition in the processing of human serum albumin.

Preferably, when obtaining recombinant human serum albumin from the transformed cells, the production method of the present invention may comprise the step of culturing the transformed cells. At this time, the process of treating a fatty acid having 16 to 22 carbon atoms or a pharmaceutically acceptable salt thereof may be achieved by adding the composition of the present invention to a culture medium for culturing the transformed cells.

The transformed cells include cells isolated from transgenic animals expressing human serum albumin as well as transformed bacteria, fungi and yeast.

Alternatively, recombinant human serum albumin is isolated and / or purified from cells isolated from cultured transformed cells or transgenic animals, from said cell culture or culture or from blood or plasma of transgenic animals, or from human plasma. After separation and / or purification of serum albumin, a step of treating the composition for thermal stabilization comprising the fatty acid of the present invention or a pharmaceutically acceptable salt thereof may be performed.

The isolated human serum albumin is human serum albumin isolated and / or purified from blood or serum of a transgenic animal expressing human serum albumin, transformed cells, culture or culture of the cells, or human blood or serum. Can be.

Methods for preparing human serum albumin preparations currently used in the art include heat treatment at 60 ° C. for 10 hours to inactivate viruses, such as AIDS and hepatitis B virus, which may be included therein.

According to a specific embodiment of the present invention, after treating various fatty acids having 16 to 22 carbon atoms in a molar ratio of 1: 1 to a physiologically active concentration such as 50 mg / ml of fatty acid free human serum albumin solution, the heat treatment is performed at a temperature of 60 ° C. or higher. As a result of confirming the degree of protein denaturation, the degree of degeneration was found to be around 70 ° C. depending on the type of fatty acid, but the rate of denaturation was reduced compared to the human serum albumin solution without the fatty acid treatment. Particularly, when oleic acid was treated, no denaturation was observed for 10 hours or more at 60 ° C., which is a heat treatment temperature used in a general method for preparing human serum albumin preparations, and it was confirmed that it was maintained without being denatured for almost 6 hours even at a higher 65 ° C. (Table 3).

In addition, as described above, the present invention is particularly heat-stabilized human serum by adding fatty acids having 16 to 22 carbon atoms before or after the concentration control, so long as the concentration of human serum albumin is maintained at a low level of 1 to 40 mg / ml. Albumin may be provided. According to one embodiment of the present invention, the heat stabilized human serum albumin may have stability capable of maintaining a secondary structure for at least 10 hours even if the heat treatment temperature is increased to 80 ° C. Moreover, even in 100 degreeC, it can maintain a stable state, without being denatured for several minutes. Therefore, in the preparation method of the human serum albumin preparation according to the present invention, the human serum albumin is controlled at a low concentration of 1 to 40 mg / ml before or after treatment with 16 to 22 carbon atoms or pharmaceutically acceptable salts thereof. Can be.

In this case, when the concentration is adjusted to 80 ° C., the time to increase the temperature to 80 ° C. is not denatured and remains stable. Therefore, when a long heat treatment process is applied, the concentration of human serum albumin is 1 to 40 mg / ml. Can be adjusted with.

Therefore, in the method for preparing human serum albumin preparation according to the present invention, when high temperature heat treatment of about 80 ° C. is required for a long time, the sample containing human serum albumin is treated with a fatty acid having 16 to 22 carbon atoms or a pharmaceutically acceptable salt thereof. Or later, to a concentration of 1 to 40 mg / ml.

As such, when the sample is adjusted to a concentration of 1 to 40 mg / ml, the sample may be heated to a temperature of 60 to 80 ° C., as described above, by formulating a thermostable human serum albumin according to the present invention to human Corresponds to the heat treatment procedure to block the possibility of contamination and / or infection for administration to.

After performing such heat treatment, the step may be concentrated to a concentration suitable for the subject, such as a concentration of 50 to 200 mg / mL, which is higher than the physiologically active concentration. This corresponds to the step of concentrating a human serum albumin sample adjusted to 1 to 40 mg / ml for a long term heat treatment to an appropriate concentration for administration to a subject including a human after heat treatment.

The concentration step may be made by a commonly used concentration method, for example, using an ultrafiltration membrane having a MWCO (Molecular Weight Cut Off) of 10 to 30 kD can be concentrated until the proper concentration by centrifugation method This is not restrictive.

An appropriate concentration of the human serum albumin preparation is not particularly limited as long as it can be used as a human serum albumin preparation, but may be 50 to 200 mg / ml.

Human serum albumin preparations prepared by such a process can be administered to a subject, including humans, by any device in which the active substance can migrate to target cells. Preferred modes of administration and preparations can be injections, such as intravenous, subcutaneous, intradermal, intramuscular, intraocular, or injectable. Injectables include aqueous solvents such as physiological saline solution and ring gel solution, vegetable oils, higher fatty acid esters (e.g., oleic acid, etc.), and non-aqueous solvents such as alcohols (e.g., ethanol, benzyl alcohol, propylene glycol, glycerin, etc.). Stabilizers (e.g., ascorbic acid, sodium bisulfite, sodium pyrosulfite, BHA, tocopherol, EDTA, etc.), emulsifiers, buffers for pH adjustment, preservatives to prevent microbial growth (Eg, phenyl mercury nitrate, chimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, and the like).

Hereinafter, the present invention will be described in more detail with reference to Examples. These examples are intended to illustrate the present invention more specifically, but the scope of the present invention is not limited by these examples.

Green serum human serum albumin was crystallized for the structural analysis of plasma-derived human serum albumin. Hereinafter, the optimum concentration for crystallization determined through the precipitant experiment was determined to be 80 mg / ml. Initial crystallization was done using screen I & II, index screen reagent, a screening kit from Hampton Research (Naguna Niguel, Ca, USA) based on sparse matrix theory (Jancarik & Kim, 1991). It was carried out by manual or automated method using drop or sitting drop vapor diffusion method. The automated method was performed using Hydra e-Drop (Thermo Scientific, Waltham, Mass., USA) used in the high-throughput crystallization system manufactured in this laboratory.

Thereafter, the crystallization process was performed at 150 mg / ml to determine the optimal concentration for crystallization of human serum albumin from Green Cross through a manual refine screen.

The final crystallization conditions of human serum albumin determined through the above procedure are as follows: 35% PEG 600, containing 0.1 M MES (2- (N-Morpholino) ethanesulfonic Acid) pH 6.5 and 0.2 M ammonium sulfate 2 μl of a hanging drop made by mixing the crystallization solution and the 150 mg / ml protein solution in a well containing 200 μl of a mother liquor, that is, a crystallization solution, was put on a cover glass. The crystals were placed by the hanging drop diffusion method.

Example 2: Screening of cryo conditions of plasma-derived human serum albumin

Finding stabilized freezing conditions is an essential step in the data collection process using Synchrotron. This is because the intensity of the radiation from the synchrotron is so strong that crystals are easily decayed during the data collection process and complete data collection is not possible. Therefore, the conditions of the crystallization solution that can flash frozen the crystals should be found.

Therefore, in the present invention, LV CryoOil (MiTeGen, Ithaca, NY) was used for human serum albumin of Green Cross. 2 μl of LV CryoOil was placed next to the drop containing Green Serum Albumin crystals of Green Cross, the crystals were transferred to the mounting loop, soaked in the LV CryoOil, and the crystals were again frozen to the mounting loop.

Example 3: X-ray Data Collection of Plasma-Derived Human Serum Albumin

Data on human serum albumin from Green Cross were collected up to 2.17 mm 3 on X-ray Australian Synchrotron. The detector was an ADSC Q315r. Each value is shown in Table 1 below. In addition, the three-dimensional structure of the serum-derived human serum albumin crystal from this is shown in FIG.

Table 1

Example 4 Solving the Phase Problem of Plasma-Derived Human Serum Albumin Using Molecular Substitution

The crystal structure of human serum albumin of Green Cross was determined by molecular replacement method. That is, when structural similarity between proteins is expected, a solution of the phase problem by molecular replacement was used using a known structure. The method finds the orientation of molecules by using a fast rotation function, such as a translation function, an R-factor search, a correlation search, and the like. The calculation finds the position of the molecule. The approximate orientations and positions thus obtained are further refined by rigid body refinement or R-factor minimization search, followed by refinement and model modification of atomic positions. rebuilding). In this study, the solution of human serum albumin (Green Cross) using EPMR using the structure of apo-human serum albumin (apo-HSA, PDB ID: 1AO6) as a search model Found.

Example 5 Identification of Three-Dimensional Crystal Structure of Plasma-Derived Human Serum Albumin

The phase of human serum albumin of Green Cross was obtained by molecular replacement method using human albumin model. Early models were created using COOT and O, which are graphic software, and energy minimization was performed using the CNS. The final model was obtained through PHENIX. Refinement values of the final model are reported in Table 2.

TABLE 2

The three-dimensional structure identified in this way is basically similar to the existing apo-HSA structure. The binding sites of octanoic acid and N-acetyl tryptophan used in the preparation of pharmaceutical grade plasma-derived HSA were also identified. Two octanates bound to sites known as fatty acid binding sites and N-acetyl tryptophan bound to Sudlow site II. In addition, the unexpected binding of fatty acids was confirmed, and after the X-ray structure and mass spectrometry analysis, it was confirmed that the fatty acids are fatty acids having 18 carbon atoms such as oleic acid. Mass spectrometry results of 20% human serum albumin injections of Green Cross and SK, which are currently commercialized, are shown in FIGS. 2 and 3, respectively.

Example 6 Effect of Fatty Acids on Thermal Degeneration of Human Serum Albumin

6.1. Effect of Oleic Acid on the Heat Degeneration of Human Serum Albumin

The purpose of this study was to investigate the effect of oleic acid found to be bound to plasma-derived human serum albumin. The present inventors predicted that the stability may be affected by temperature. Therefore, after adding oleic acid to a fatty acid free human serum albumin (A3782, Sigma-Aldrich) solution and performing a heat treatment experiment at a high temperature, the changes occurring are observed and the results are shown in Tables 3 to 6 and FIG. 4 is shown. As a control, a fatty acid free human serum albumin solution without oleic acid was used. The concentration of the human serum albumin solution was 50 mg / ml, 25 μl of the solution was added, and oleic acid was added to the experimental group in the same molar ratio. Specifically, the temperature was maintained at 65 ° C., 70 ° C., and 75 ° C. to determine whether precipitates were formed due to protein denaturation at 30, 5, and 3 minute intervals, respectively. At 75 ° C, the highest temperature, no difference was observed between the control and experimental groups. However, in the control group, precipitation began to occur after 10 minutes at 70 ° C. and after 30 minutes at 65 ° C., whereas precipitation began to form after 30 minutes at 70 ° C. and after 330 minutes at 65 ° C. in the oleic acid-added group. Therefore, it was confirmed that oleic acid can contribute significantly to the stabilization of human serum albumin at high temperature. That is, since the addition of oleic acid can give stability to the heat treatment process, which is an essential process for the formulation, it was confirmed that it can be used in place of sodium octanate and N-acetyl tryptophan, which was added as a conventional heat stabilizer.

TABLE 3

Table 4

Table 5

Table 6

6.2. Effects of Various C18 Fatty Acids on Thermal Degeneration of Human Serum Albumin

In the same manner as the experiment that confirmed the stability to the denaturation by heat treatment using the oleic acid in Example 6.1. 70 ℃ using oleic acid as well as other C18 fatty acids stearic acid, linoleic acid, α- linolenic acid and γ- linolenic acid The thermal stabilization effect was confirmed at. Up to 30 minutes were observed at 3 minute intervals and thereafter at 5 minute intervals. As a control, a fatty acid free human serum albumin solution which was not treated with fatty acids was used as in Example 6.1. As shown in Table 7 below, it was confirmed that all of the experimental groups treated with the C18 fatty acid had stability against denaturation by excellent heat treatment compared to the control human serum albumin. As described in Example 6.1 above, each fatty acid was added to have the same molar ratio as albumin.

TABLE 7

6.3. Effects of Fatty Acids of Various Carbon Numbers 16 on Thermal Degeneration of Human Serum Albumin

Example 6.1 above. In the same manner as in the experiment to confirm the stability of the denaturation by heat treatment using a variety of carbon 18 fatty acids, including oleic acid and in 6.2. Palmitic acid (palmitic acid) and palmitol as fatty acids of 16 carbon atoms as well as oleic acid of 18 carbon atoms The thermal stabilization effect was confirmed at 65 ° C. using lemic acid. Samples were warmed to 65 ° C. and observed at 5 minute intervals. Example 6.1 as a control. And as in 6.2, a fatty acid free human serum albumin solution which was not treated with fatty acids was used. As shown in Table 8 below, it was confirmed that all of the experimental groups treated with the C16 fatty acid had stability against denaturation by heat treatment significantly superior to the control human serum albumin. Example 6.1 above. And each fatty acid was added to have the same molar ratio as albumin.

Table 8

6.4. Effect of EPA and DHA on Heat Degeneration of Human Serum Albumin

Example 6.1 above. EPA (eicosapentaenoic acid) and DHA (docosahexanoic), which are fatty acids having 20 and 22 carbon atoms, as well as oleic acid having 18 carbon atoms, in the same manner as in the experiments that confirmed the stability to degeneration by heat treatment using various fatty acids having 16 and 18 carbon atoms in 6.3. acid) was used to determine the thermal stabilization effect at 65 ℃. The samples were warmed to 65 ° C. and observed at 5 minute intervals for the first 60 minutes, then at 10 minute intervals up to 130 minutes, and then at 30 minute intervals thereafter. Example 6.1 as a control. Fatty acid free human serum albumin solution without fatty acid treatment was used as in 6.3. As shown in the following Table 9, it was confirmed that all of the experimental group treated with EPA and DHA had a stability against denaturation by heat treatment at a level similar to oleic acid, which is significantly superior to the control human serum albumin. Example 6.1 above. Each fatty acid was added to have the same molar ratio as albumin as described in 6.3.

Table 9

Example 7: Structural Stability Test of Heat Treatment of Low Concentration Human Serum Albumin Sample

(1) Preparation of human serum albumin sample

Green cross albumin, A3782 albumin (Sigma) and A1653 albumin (Sigma) were diluted with 20 mM potassium phosphate buffer (pH 7.5) to 1 mg / ml, respectively. And oleic acid was added to green cross albumin and A3782 albumin so that albumin and molar ratio was 1: 1, and then reacted overnight at 4 ° C. to prepare a total of five human serum albumin samples; i) Green cross (room temperature), ii) A1653 albumin, iii) A3782 albumin, iv) A3782 albumin + oleic acid (1: 1 molar ratio), v) Green cross albumin + oleic acid (1: 1 molar ratio).

(2) Measurement method of circular dichroism of albumin

Circular dichroism can predict the composition ratio of the secondary structure of the protein and can measure the structural change of the heat denatured protein. The original non-thermally denatured green cross albumin and all five thermally denatured albumin samples were diluted with 20 mM potassium phosphate buffer (pH 7.5) to 0.25 mg / ml.

The circular dichroism was measured using a Jasco J-715 spectropolarimeter with a quartz cuvette with a path length of 0.1 cm and measured three times with a data pitch of 0.2 nm, band width of 1.0 nm, response time of 4 seconds, and scanning speed of 50 nm / min. It was. From the measured circular dichroism measurements, the composition ratio of the secondary structure was predicted using the SELCON3 program included in the Dicroprot program.

(3) Structural stability test for heat treatment of human serum albumin at 60 ° C

Five human serum albumin samples (green cross albumin, A3782 albumin, A1653 albumin, green cross albumin + oleic acid (1: 1 molar ratio), A3782 albumin + oleic acid (1: 1 molar ratio) prepared in Example 7 (1) were 60 ° C. Heated at 10 h.

Then, as a result of measuring the circular dichroism by the method of Example 7 (2), the composition ratio of the secondary structure of the albumin sample was as shown in Table 10 below.

Table 10

	α-Helix	β-sheet	Turn	poly (Pro) Ⅱ	Unordered
Green Cross (room temperature)	0.706	0.038	0.070	0.022	0.193
A1653 albumin	0.481	0.072	0.105	0.042	0.311
A3782 albumin	0.308	0.130	0.133	0.052	0.379
Green Cross Albumin	0.514	0.071	0.109	0.029	0.302
A3782 albumin + oleic acid (1: 1)	0.514	0.079	0.104	0.030	0.283
Green Cross Albumin + Oleic Acid (1: 1)	0.740	0.011	0.096	0.013	0.151

As a result, as shown in Table 10, the ratio of the α-helix of the thermally denatured specimens is generally reduced, and the unordered structure is increased compared to the green cross (normal temperature) specimen that does not denature. Showed results.

However, the green cross albumin + oleic acid (1: 1) sample was found to be very stable even when heated at 60 ° C for 10 hours because the ratio was similar to that of the green cross (room temperature) sample. In addition, the A3782 albumin + oleic acid (1: 1) samples also showed higher thermal stability due to a higher ratio of α-helix (α-Helix) after heat denaturation compared to A3782 albumin. This suggests that thermal stability increases when albumin reacts with oleic acid.

(4) Structural Stability Test of Heat Treatment of Human Serum Albumin at 70 ° C

Five human serum albumin samples (Green Cross Albumin, A3782 Albumin, A1653 Albumin, Green Cross Albumin + Oleic Acid (1: 1 molar ratio), A3782 Albumin + Oleic Acid (1: 1 molar ratio) prepared in Example 7 (1) were prepared at 70 ° C. Heated at 10 h.

Then, as a result of measuring the circular dichroism by the method of 7 (2), the composition ratio of the secondary structure of the albumin sample was as shown in Table 11.

Table 11

	α-Helix	β-sheet	Turn	poly (Pro) Ⅱ	Unordered
Green Cross (room temperature)	0.706	0.038	0.070	0.022	0.193
A1653 albumin	0.330	0.124	0.144	0.055	0.369
A3782 albumin	0.309	0.107	0.141	0.053	0.378
Green Cross Albumin	0.441	0.022	0.207	0.022	0.314
A3782 albumin + oleic acid (1: 1)	0.724	0.017	0.079	0.030	0.163
Green Cross Albumin + Oleic Acid (1: 1)	0.627	0.036	0.081	0.030	0.234

As shown in Table 11, as a result of the heat treatment as described above, the ratio of α-helix (α-Helix) of the thermally denatured specimens is generally reduced and disorderly compared to the green cross (room temperature) specimen without thermal denaturation. (unordered) structure increased.

However, the green cross albumin + oleic acid (1: 1 molar ratio) sample and the A3782 albumin + oleic acid (1: 1 molar ratio) sample subjected to heat treatment after oleic acid reaction had a ratio of α-helix (α-Helix) to green cross (room temperature) sample. Similar to that of the albumin which did not react with oleic acid was confirmed to be very stable even when heated at 70 ℃ for 10 hours.

(5) Structural stability test for heat treatment of human serum albumin at 80 ° C

Five human serum albumin samples (Green Cross Albumin, A3782 Albumin, A1653 Albumin, Green Cross Albumin + Oleic Acid (1: 1 molar ratio), A3782 Albumin + Oleic Acid (1: 1 molar ratio) prepared in Example 7 (1) were prepared at 80 ° C. Heated at 10 h.

Then, as a result of measuring the circular dichroism by the method of 7 (2), the composition ratio of the secondary structure of the albumin sample was as shown in Table 12 below.

Table 12

	α-Helix	β-sheet	Turn	poly (Pro) Ⅱ	Unordered
Green Cross (room temperature)	0.706	0.038	0.070	0.022	0.193
A1653 albumin	0.270	0.127	0.149	0.054	0.390
A3782 albumin	0.217	0.143	0.178	0.043	0.432
Green Cross Albumin	0.298	0.135	0.131	0.064	0.380
A3782 albumin + oleic acid (1: 1)	0.212	0.169	0.162	0.066	0.404
Green Cross Albumin + Oleic Acid (1: 1)	0.132	0.223	0.188	0.061	0.404

As shown in Table 12, as a result of the heat treatment, the ratio of α-helix (α-Helix) of all thermally denatured specimens was reduced compared to the non-thermally denatured green cross (normal temperature) specimens, and the order was unordered. Increased.

However, unlike at 60 ℃ and 70 ℃, the ratio of α-helix (α-Helix) decreased and unordered in both green cross albumin + oleic acid (1: 1 molar ratio) sample and A3782 albumin + oleic acid (1: 1 molar ratio). ) Structure increased.

Example 8 Concentration Range Test of Albumin Maintaining Thermal Stability

20 mM potassium phosphate to make A3782 albumin (Sigma) 1 mg / ml, 5mg / ml, 10mg / ml, 15mg / ml, 20mg / ml, 25mg / ml, 30mg / ml, 35mg / ml, 40mg / ml, 45mg / ml Diluted with pH 7.5 buffer. And oleic acid was added to A3782 albumin so as to have a molar ratio of albumin 1: 1 and then reacted overnight at 4 ° C.

Then, 1 mg / ml A3782 albumin + oleic acid (1: 1 molar ratio), 5 mg / ml A3782 albumin + oleic acid (1: 1 molar ratio), 10 mg / ml A3782 albumin + oleic acid (1: 1 molar ratio), 15 mg / ml A3782 albumin + Oleic acid (1: 1 molar ratio), 20 mg / ml A3782 albumin + oleic acid (1: 1 molar ratio), 25 mg / ml A3782 albumin + oleic acid (1: 1 molar ratio), 30 mg / ml A3782 albumin + oleic acid (1: 1 molar ratio), Ten kinds of specimens, such as 35mg / ml A3782 albumin + oleic acid (1: 1 molar ratio), 40mg / ml A3782 albumin + oleic acid (1: 1 molar ratio), 45mg / ml A3782 albumin + oleic acid (1: 1 molar ratio) Heated at 10 h.

Circular dichroism of albumin was measured according to the method of Example 7 (2), and as a result, a sample of 1 mg / ml A3782 albumin + oleic acid (1: 1 molar ratio) was not denatured green cross. And the ratio of α-helix (α-Helix) was similar (Table 11 and Figure 6). 1-40 mg / ml of A3782 albumin + oleic acid (1: 1 molar ratio) in the dichroic polarization graph of the thermally denatured 1-45 mg / ml A3782 albumin + oleic acid (1: 1 molar ratio) sample was 1 mg / ml A3782 Albumin + oleic acid (1: 1 molar ratio) and its spectrum was similar to confirm that the composition ratio of the protein secondary structure did not change significantly (Fig. 7).

On the other hand, the 45 mg / ml A3782 albumin + oleic acid (1: 1) sample significantly weakened the dichroic polarization signal, which lowered the ratio of α-Helix and increased the unordered structure. Therefore, the A3782 albumin + oleic acid (1: 1 molar ratio) specimen was confirmed that the high thermal stability is maintained in the concentration range of 1 to 40 mg / ㎖ (Fig. 8).

Example 9 Evaluation of Thermal Stability of Albumin with Addition of Unsaturated Fatty Acids of 16 to 22

A3782 albumin (Sigma) was diluted with 20 mM potassium phosphate pH 7.5 buffer to 1 mg / ml. Then, oleic acid, linoleic acid, α-linolenic acid, γ-linolenic acid and palmitoleic acid were added to A3782 albumin so that the molar ratio of albumin was 1: 1 and then reacted at 4 ° C. overnight.

A3782 albumin + oleic acid (1: 1 molar ratio), A3782 albumin + linoleic acid (1: 1 molar ratio), A3782 albumin + α-linolenic acid (1: 1 molar ratio), A3782 albumin + γ-linolenic acid (1: 1 molar ratio), A3782 albumin Five specimens were heated at 70 ° C. for 10 hours, such as + palmitoleic acid (1: 1 molar ratio).

Circular dichroism of albumin was measured according to the method of Example 7 (2). As a result, the samples of A3782 albumin + oleic acid (1: 1 molar ratio) were not denatured and the green cross specimen was α-. The ratio of the helix structure (α-Helix) was similar (Table 11 and Figure 6). In the circular dichroism graph of the albumin sample containing 16 to 24 unsaturated fatty acids, the sample containing the unsaturated fatty acid having 16 to 22 carbon atoms except for oleic acid was generally different from the sample containing oleic acid and the circular dichroism spectrum. Similarly it was confirmed that there is no significant change in the secondary structure of the protein (Fig. 9).

These results suggest that the same or corresponding effects can be obtained by using fatty acids having 16 to 22 carbon atoms in addition to oleic acid in the method of the present invention.

From the above description, those skilled in the art will appreciate that the present invention can be implemented in other specific forms without changing the technical spirit or essential features. In this regard, the examples described above are illustrative in all respects and should be understood as not limiting. The scope of the present invention should be construed that all changes or modifications derived from the meaning and scope of the following claims and equivalent concepts rather than the detailed description are included in the scope of the present invention.

Claims (18)

1. A composition for thermal stabilization for preparing a human serum albumin preparation comprising a fatty acid selected from the group consisting of fatty acids having 16 to 22 carbon atoms or a pharmaceutically acceptable salt thereof.
2. The method of claim 1,

   Wherein said human serum albumin is human plasma-derived or recombinant human serum albumin.
3. The method of claim 1,

   The fatty acid may be palmitic acid, palmitoleic acid, stearic acid (octadecanoic acid), oleic acid, linoleic acid, linolenic acid, EPA (eicosapentaenoic) acid) and DHA (docosahexaenoic acid).
4. The method of claim 1,

   The salt of the fatty acid is an alkali metal or alkaline earth metal salt.
5. The method of claim 1,

   Wherein said fatty acid is added in a molar ratio of 1: 0.5 to 1: 2 relative to albumin.
6. A first step of adjusting the concentration of human serum albumin to 1 to 40 mg / ml; And

   A method for producing thermostable human serum albumin comprising the step of adding a fatty acid selected from the group consisting of fatty acids having 16 to 22 carbon atoms or a pharmaceutically acceptable salt thereof to human serum albumin.
7. The method of claim 6,

   The first and second steps are carried out in the order or in the reverse order to prepare a thermostable human serum albumin.
8. The method of claim 6,

   The fatty acid may be palmitic acid, palmitoleic acid, stearic acid (octadecanoic acid), oleic acid, linoleic acid, linolenic acid, EPA (eicosapentaenoic) acid) and DHA (docosahwxaenoic acid) is a method for producing thermostable human serum albumin which is at least one fatty acid selected from the group consisting of.
9. The method of claim 6,

   When the heat-stabilized human serum albumin is heat treated at a temperature of 60 to 80 ° C. for 6 to 15 hours, α-helical structure (α-Helix) is maintained at 80% or more, heat stabilized human serum albumin Manufacturing method.
10. The method of claim 6,

    The fatty acid or a pharmaceutically acceptable salt thereof is added in a molar ratio of 1: 0.5 to 1: 2 relative to human serum albumin.
11. In the method for producing a human serum albumin preparation,

    A method for producing a human serum albumin preparation comprising the step of treating human serum albumin with a fatty acid or a pharmaceutically acceptable salt thereof selected from the group consisting of fatty acids having 16 to 22 carbon atoms.
12. The method of claim 11,

    The human serum albumin is human plasma-derived or recombinant human serum albumin is a method for producing a human serum albumin preparation.
13. The method of claim 11,

    The human serum albumin is prepared by the method comprising the step of culturing the transformed cells expressing human serum albumin having the amino acid sequence of SEQ ID NO: 1.
14. The method of claim 11,

    A method for producing a human serum albumin preparation, characterized by adding a fatty acid selected from the group consisting of fatty acids having 16 to 22 carbon atoms to the isolated human serum albumin.
15. The method of claim 14,

    The isolated human serum albumin is a human serum albumin preparation isolated from blood or serum of a transgenic animal expressing human serum albumin, or transformed cells, culture or culture of the cells, or human blood or serum. Manufacturing method.
16. The method of claim 11,

    The human serum albumin is adjusted to a concentration of 1 to 40 mg / ㎖ before or after the treatment of a fatty acid or a pharmaceutically acceptable salt thereof selected from the group consisting of fatty acids having 16 to 22 carbon atoms to prepare a human serum albumin preparation Way.
17. The method of claim 16,

    The heat treatment is to be carried out at 60 to 80 ℃, method for preparing human serum albumin.
18. The method of claim 17,

    The method further comprises the step of concentrating the heat-treated human serum albumin to a concentration of 50 to 200 mg / ㎖, human serum albumin preparation method.

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