KR101568360B1 - Method for preparing thermal-stabilized human serum albumin - Google Patents

Abstract

The present invention relates to a method for the treatment of human serum albumin, comprising the step of treating a fatty acid selected from the group consisting of fatty acids having from 16 to 24 carbon atoms or a pharmaceutically acceptable salt thereof with human serum albumin at a concentration of 1 to 40 mg / And a method for producing the same.

Description

[0001] The present invention relates to a method for preparing thermostabilized human serum albumin,

The present invention relates to a method for the treatment of human serum albumin, comprising the step of treating a fatty acid selected from the group consisting of fatty acids having from 16 to 24 carbon atoms or a pharmaceutically acceptable salt thereof with human serum albumin at a concentration of 1 to 40 mg / And a method for producing the same.

Albumin is a very abundant protein in the blood, produced in the liver. Structurally, human serum albumin (HSA) is composed of 585 amino acids (66,438 Da) and consists of 17 disulfide bridges and one free cysteine (Cys34). In addition, albumin plays a role of maintaining and restoring plasma volume, the role of regulating and transferring colloid osmotic pressure of blood by binding with various ligands such as water, calcium, sodium, potassium, cation, fatty acid, hormone, bilirubin, It acts as an amino acid source in malnutrition. In particular, the binding of the drug to albumin is highly involved in the drug efficacy of the drug. On the other hand, purified HSA is used for the treatment of hypoalbuminemia due to, for example, surgical operation, hemorrhagic shock or burn and albumin synthesis dysfunction such as nephrotic syndromes. In addition, albumin is used as a supplement to bring about higher eukaryotic cell growth, or as a pharmaceutical additive in therapeutic protein formulation. Another known function of serum albumin is the action of an oxygen carrier that adsorbs and desorbs oxygen with oxygen partial pressure similar to hemoglobin. Therefore, it can be administered instead of hemoglobin in an emergency. At present, the demand for albumin products is being supplemented by albumin extracted from human blood.

Conventionally, HSA was prepared by various methods of purification after obtaining the fraction containing HSA (fraction V) by low temperature ethanol fractionation of Cone from human plasma or a method similar thereto. Examples of the purification method include a purification method commonly used in protein chemistry such as salting out method, ultrafiltration method, isoelectric point precipitation method, electrophoresis method, ion exchange chromatography method, gel filtration chromatography method, Affinity chromatography and the like can be used. In the purification process of human serum albumin, since many kinds of contaminants derived from biological tissue, cells, blood, etc. are mixed in the sample obtained from plasma, purification of human serum albumin needs to be used in combination with the above methods have. However, the amount of albumin purified from human plasma is limited by this process. Therefore, recently, genetic manipulation techniques have been applied to a raw material in a way that does not depend on human plasma, and yeast and Bacillus using subtilis) has been developed a method for producing human serum albumin.

Typically, albumin preparations in the form of aqueous solutions are subjected to a heat treatment at 60 占 폚 for 10 hours to inactivate viruses that may contaminate the preparation. The industrial production of albumin is also carried out under conditions different from those in the human body. Therefore, in view of the characteristics of albumin which is easily denatured and aggregated by heat or the like, the above process is highly likely to be denatured or form a multimer during the course of the process. At present, commercially available 5% or 20% albumin preparations are widely used without any adverse side effects, and thus it is considered that the multimers contained in the preparation are harmless to human body. However, the increase of blood pressure, fever, tachycardia, tremor, , Chills and other side effects are reported. Therefore, it is necessary to develop a process that does not denature albumin in the heat treatment process.

Accordingly, the present inventors have made intensive efforts to develop an optimal process capable of maintaining high stability of human serum albumin even in a long heat treatment process. As a result, the inventors have found that a salt can be obtained by adding a salt of human serum albumin having a concentration of 1 to 40 mg / It is confirmed that the stability can be maintained at a high level even for a long time heat treatment for about 10 hours when the heat is applied to include the fatty acid, thereby completing the present invention.

It is an object of the present invention to provide a method for treating human serum albumin which comprises treating a fatty acid selected from the group consisting of fatty acids having 16 to 24 carbon atoms or a pharmaceutically acceptable salt thereof to human serum albumin at a concentration of 1 to 40 mg / And a method for producing stabilized human serum albumin.

Another object of the present invention is to provide a method for the preparation of a pharmaceutical composition which comprises (a) treating human serum albumin with a fatty acid selected from the group consisting of fatty acids having from 16 to 24 carbon atoms or a pharmaceutically acceptable salt thereof; (b) adjusting the concentration of the human serum albumin to a concentration of 1 to 40 mg / ml. The present invention also provides a method for producing heat-stabilized human serum albumin.

As an embodiment for achieving the above object, the present invention includes a step of treating a fatty acid or a pharmaceutically acceptable salt thereof selected from the group consisting of fatty acids having 16 to 24 carbon atoms to human serum albumin at a concentration of 1 to 40 mg / ml And a method for producing heat stabilized human serum albumin.

According to the preparation method of the present invention, by treating a human serum albumin at a concentration of 1 to 40 mg / ml with a fatty acid selected from the group consisting of fatty acids having 16 to 24 carbon atoms or a pharmaceutically acceptable salt thereof, Albumin can be produced.

Previously, the adverse effects of albumin on infectious diseases were reported, and as a method to overcome this, heat treatment process was introduced at 60 ° C for 10 hours together with virus killing effect by cold ethanol. However, even though albumin is a structurally very stable protein, the formation of dimers, oligomers and polymers in the heat treatment process can be induced, and gelation phenomenon has been reported in severe cases. Therefore, in the production of a human serum albumin preparation, it is important to develop a method capable of maintaining the stability of human serum albumin for a long time in a heat treatment step required. Accordingly, the present invention provides a method of treating a human serum albumin having a low concentration of 1 to 40 mg / ml with a fatty acid having a carbon number of 16 to 24 or a pharmaceutically acceptable salt thereof at a temperature of 60 to 80 캜 for 10 hours or more And the secondary structure of albumin was maintained. The method of the present invention can provide the stability of the albumin structure even in the heat treatment process for several hours, and thus can be useful for the development of the albumin preparation with increased stability.

In the present invention, the term "albumin" is one of the proteins constituting the basic substance of a cell. It exists in the blood and is produced in the liver. Among the simple proteins present in the natural state, the molecular weight is the smallest. Serum albumin in blood has a function of maintaining and restoring plasma volume, preventing shock due to excessive bleeding, and being used for surgery and burn treatment. It is also known to have oxygen transfer capability similar to hemoglobin. For the purpose of the present invention, the albumin should be provided with stability against heat treatment in the process of formulating it. The albumin to be subjected to the heat treatment may include all albumin which can be formulated. Preferably, Or recombinant human serum albumin produced by genetic engineering, more preferably human serum albumin derived from human plasma, but is not limited thereto.

The term " recombinant human serum albumin " in the present invention refers to human serum albumin artificially produced as a concept to be compared with natural human serum albumin derived from human plasma. For example, human serum albumin obtained from a transformant prepared to express a polypeptide having the amino acid sequence of SEQ ID NO: 1 (sequence of HSA), may have the same amino acid sequence as human serum albumin separated from plasma, It does not.

As used herein, the term "transformant" is a genetically modified organism which is genetically modified to express a specific gene through genetic engineering. For the purpose of the present invention, May be a polypeptide having the amino acid sequence of SEQ ID NO: 1, for example, a gene encoding human serum albumin. Preferably, the genetically modified organism may be, but is not limited to, cells or transgenic animals such as transformed bacteria, fungi and yeast. Preferably, the transgenic animal may be a mammal such as a mouse, a rat, or a guinea pig, but is not limited thereto.

The thermally stabilized human serum albumin according to the production method of the present invention can be formulated into human serum albumin and administered to an individual including human. At this time, the human serum albumin preparation may include a commercially available albumin preparation, and may also include a preparation having similar components, functions and effects as those of a commercially available albumin preparation, which is prepared so as to be substituted therewith. In the case of commercially available albumin preparations, there is a problem of dependence on the blood of a limited supply source. To solve this problem, a recombinant human serum albumin preparation using recombinant human serum albumin can be produced.

In order to prepare the heat stabilized human serum albumin as a preparation that can be administered to the human body, a heat treatment process is required to prevent virus contamination and / or other infections. However, similar to ordinary proteins, human serum albumin can cause denaturation such as structural change or agglomeration at high temperature. Therefore, it is necessary to add an additive to stabilize it. Currently, octanoate such as sodium octanoate and N-acetyl tryptophan are used in the production process of plasma-derived albumin preparation. However, these substances are substances that are originally added to improve the stability of the heat treatment process rather than substances existing in the blood plasma, and unlike natural human serum albumin, when they are administered into the human body, .

Therefore, it is necessary to maintain the form and structure most similar to natural human serum albumin when producing human plasma-derived or recombinant human serum albumin preparations, if possible. Since the recombinant human serum albumin has some properties different from those of human serum albumin derived from human plasma, it is desirable to treat the substance present in the living body as a composition for thermal stabilization for preparing a recombinant human serum albumin preparation, It can be safer. Thus, in the present invention, a fatty acid having 16 to 24 carbon atoms identified as being bound to human serum albumin separated from plasma was used.

The term "fatty acid having 16 to 24 carbon atoms" in the present invention means a fatty acid having 16 to 24 carbons in a fatty acid. The term " fatty acid having 16 to 24 carbon atoms " includes hydrophilic carboxyl group at one end and hydrophobic carbon chain at the other end. In this case, the type of the carbon chain may vary depending on the number and position of the double bonds, and preferably palmitic acid, palmitoleic acid, fatty acid of carbon number 16, stearic acid oleic acid, linoleic acid and linolenic acid, eicosapentaenoic acid (EPA), which is a fatty acid having 20 carbon atoms, and docosahexaenoic acid, which is a fatty acid having 22 carbon atoms, as well as fatty acids such as stearic acid, octadecanoic acid, oleic acid, linoleic acid and linolenic acid, Hexaenoic acid (DHA), or a combination thereof, but is not limited thereto. The fatty acid may also be used in the form of a pharmaceutically acceptable salt.

In the present invention, the term "palmitic acid" is a saturated fatty acid having 16 carbon chains and is referred to as hexadecanoic acid under the IUPAC name. CH ₃ (CH ₂ ) ₁₄ CO ₂ H and is the most common fatty acid found in plants, animals and microorganisms. It is also present in the major components of palm oil, meat, cheese, butter and dairy products. The salt and ester form of palmitic acid is called palmitate. It is present in the form of palmitate anion at basic pH. In the body, excess carbohydrates are converted to palmitic acid. Palmitoic acid is the first fatty acid produced during fatty acid synthesis and is a precursor of long chain fatty acids. Biologically, some proteins can be modified by binding to palmitoyl groups, which is called palmitoylation, which is important for membrane localization of proteins.

The term "palmitoleic acid" in the present invention is also referred to as omega-7 monounsaturated fatty acid, cis-palmitoleic acid, 9-cishexadecenoic acid, IUPAC The name is hexadec-9-enoic acid. It has the chemical formula CH ₃ (CH ₂ ) ₅ CH = CH (CH ₂ ) ₇ CO ₂ H as the fatty acid of carbon 16 containing one double bond and is a component of the glyceride of human adipose tissue. It is present in all tissues and is found mainly in liver at high concentration. It is biosynthesized from palmitic acid by the action of delta-9 desaturase. It is a beneficial fatty acid that not only inhibits the destruction of insulin-secreting beta cells but also inhibits inflammation and increases insulin sensitivity. Edible palmitoleic acid can be extracted from plants, animals and marine organism oils.

In the present invention, the term "stearic acid" is a saturated fatty acid having 18 carbon chains and is referred to as octadecanoic acid under the IUPAC name. Is a waxy solid having the formula CH ₃ (CH ₂ ) ₁₆ CO ₂ H. Salts and esters thereof are referred to as stearates. In addition to palmitic acid, stearic acid is one of the most widely available 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. Because of this property, it can be used as a surfactant or softener.

The term "oleic acid" in the present invention is a colorless and odorless oil produced from the retention of various plants and animals. Commercially available products can also be yellow. It is chemically classified as monounsaturated omega-9 fatty acid and has the chemical formula CH ₃ (CH ₂ ) ₇ CH═CH (CH ₂ ) ₇ CO ₂ H. On the other hand, oleic acid is the most abundant fatty acid in human adipose tissue.

The term "linoleic acid" in the present invention is an unsaturated n-6 fatty acid and a colorless liquid at room temperature. The linoleic acid is one of the essential fatty acids that can not be synthesized from other food ingredients in the body. It is a polyunsaturated fatty acid used especially for the biosynthesis of arachidonic acid and some prostaglandins. It is found in cell membrane lipids and is abundant in vegetable oils. Chemically a chain of 18 carbons and a carboxylic acid with two cis double bonds, the first double bond is located at the 6th carbon from the methyl end, which is the hydrophobic end.

In the present invention, the term "linolenic acid" refers to linolenic acid in two forms, alpha and gamma, or a mixture thereof. The linolenic acid is usually in the form of a linolenate and is present in vegetable oils. Linolenic acid is a carboxylic acid having 18 carbon chains and 3 cysteine double bonds, the first double bond being a polyunsaturated n-3 fatty acid located at the methyl terminus, i.e., the third carbon from the n-terminus, -9,12,15-octadecatrienoic acid (all- cis- 9,12,15-octadecatrienoic acid). Linolenic acid which is an isomer thereof is a polyunsaturated n-6 fatty acid having the same number of carbon atoms as the? -Linolenic acid and having the same number of double bonds but the first double bond being located at the 6th carbon from the n- , The positions of the double bonds in the chain are different from each other. γ-linolenic acid, also called gamolenic acid, is known to exert its effects on inflammatory and autoimmune diseases.

The term "EPA (eicosapentaenoic acid)" in the present invention is an omega-3 fatty acid, also called timnodonic acid, and its chemical structure is a carboxylic acid having five cis double bonds in 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 an eicosanoid, prostaglandin-3, thromboxane-3 and leukotriene-5 Lt; / RTI > precursor. It is abundant in seaweeds and fish, especially fish oil, and is also present in human breast milk. Fish do not naturally produce EPA, but are obtained from the algae they consume. It is possible to convert alpha-linolenic acid to EPA in the human body, but the efficiency is very low. On the other hand, EPA acts as a precursor of DHA.

The term "DHA (docosahexaenoic acid)" in the present invention is an omega-3 fatty acid which is also a primary component of the human brain, cerebral cortex, skin, sperm, testis and retina. Alpha-linolenic acid, or may be obtained directly from breast milk or fish oil. The chemical structure is a carboxylic acid having 6 cis double bonds in 22 carbon chains. The first double bond is located at the omega end, the third carbon from the hydrocarbon end. (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 concentrated in the organisms located in the upper part of the food chain, originating from photosynthesis and microalgae of the taga nutrition organism. Can be produced by the conversion of alpha-linolenic acid in the body, and the conversion rate is 15% higher in females and the conversion to testosterone or inhibition of estradiol conversion to estradiol decreases the conversion to DHA. Sperm, brain phospholipid and the main fatty acids of the retina, DHA intake can reduce the risk of heart disease by lowering blood triglyceride levels. Also, levels of DHA below normal levels are associated with Alzheimer's.

The fatty acids may be labeled N: X (n-Y) by notation according to lipid number. N represents the number of carbon atoms of the entire chain, that is, 16, 18, 20 or 22, X represents the number of double bonds contained in the chain, and Y represents the position of carbon in which the double bond starts from the methyl end of each fatty acid. We write ω to the n-dash. For example, the palmitic acid of the present invention has a 16: 1 (n-7) or 16: 1 (ω- 7), since stearic acid is a saturated fatty acid having 18 carbon atoms and 18: 0 oleic acid has one double bond from the methyl end to the 9th carbon, the ratio of 18: 1 (n-9) 18: 3 (n-6) or 18: 2 (omega-6), linolenic acid-18: 3 6) or 18: 3 (ω-6), EPA 20: 5 (n-3) and finally DHA 22: 6 (n-3).

The term "pharmaceutically acceptable salt" as used herein means a salt having a desired biological and / or physiological activity of the heat-stabilized human serum albumin, which is not toxic to cells or humans exposed to the human serum albumin, Undesirable toxicological effects mean all salts that exhibit minimal. Salts are useful as acid addition salts formed by pharmaceutically acceptable free acids. The acid addition salt is prepared by a conventional method, for example, by dissolving the compound in an excess amount of an acid aqueous solution, and precipitating the salt using a water-miscible organic solvent such as methanol, ethanol, acetone or acetonitrile. The same molar amount of the compound and the acid or alcohol (e.g., glycol monomethyl ether) in water may be heated and then the mixture may be evaporated to dryness, or the precipitated salt may be subjected to suction filtration. As the free acid, inorganic acid and organic acid can be used. As the inorganic acid, hydrochloric acid, hydrobromic acid, phosphoric acid, nitric acid, sulfuric acid, tartaric acid and the like can be used. Examples of the organic acid include methanesulfonic acid, p-toluenesulfonic acid, acetic acid, Maleic acid, succinic acid, oxalic acid, benzoic acid, tartaric acid, fumaric acid, mandelic 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, But are not limited to these.

In addition, preferably a base can be used to make a pharmaceutically acceptable metal salt. The alkali metal or alkaline earth metal salt is obtained, for example, by dissolving the compound in an excess amount of an alkali metal hydroxide or alkaline earth metal hydroxide solution, filtering the non-soluble compound salt, and evaporating and drying the filtrate. At this time, as the metal salt, it is preferable to produce sodium, potassium or calcium salt particularly, but not limited thereto. The corresponding silver salt can also be obtained by reacting an alkali metal or alkaline earth metal salt with a suitable silver salt (e.g., silver nitrate).

The fatty acid having 16 to 24 carbon atoms or a pharmaceutically acceptable salt thereof includes salts of acidic or basic groups which may exist in fatty acids having 16 to 24 carbon atoms, unless otherwise indicated. For example, pharmaceutically acceptable salts may include sodium, calcium and potassium salts of hydroxy groups, and other pharmaceutically acceptable salts of amino groups include hydrobromide, sulfate, hydrogen sulfate, phosphate, hydrogen phosphate, (Mesylate), p-toluenesulfonate (tosylate) salts, and the like, which are known in the art and which can be prepared by methods known to those skilled in the art, such as hydrogen phosphate, acetate, succinate, citrate, tartrate, lactate, mandelate, methanesulfonate . Preferably, it may be an alkali metal or an alkaline earth metal salt, but is not limited thereto.

As used herein, the term "treatment" refers collectively to all processes involving the chemical or biological action of a fatty acid having from 16 to 24 carbon atoms or a pharmaceutically acceptable salt thereof for thermal stabilization. For example, it can be applied at any stage before the heat treatment, such as by incorporating it into the culture medium in the course of culturing for the preparation of thermally stabilized human serum albumin, or by incorporating it in the form of a composition during the processing of human serum albumin.

Preferably, the heat stabilized human serum albumin of the present invention can be treated to have a molar ratio of the fatty acid or its pharmaceutically acceptable salt to albumin in a molar ratio of 1: 0.5 to 1: 2, preferably 1: 0.7 to 1: 1: 1.5, more preferably 1: 0.8 to 1: 1.2, and most preferably 1: 1.

In the production method of the present invention, the fatty acid having 16 to 24 carbon atoms or a pharmaceutically acceptable salt thereof is treated with 1 to 40 mg / ml of human serum albumin. When the above-mentioned fatty acids having a carbon number of 16 to 24 are treated with low-concentration human serum albumin at a concentration of 1 to 40 mg / ml and heat is applied thereto for 5 to 16 hours, 80-80 of the? -Helical structure of human serum albumin To 100%.

At this time, in order to adjust the concentration of the human serum albumin to 1 to 40 mg / ml, various dilution buffers generally used in the production of albumin preparations may be used. Examples of such dilute buffers may include histidine-buffer, citrate-buffer, succinate-acetate, acetate-buffer or phosphate-buffer, preferably 50 mM potassium phosphate and 150 mM NaCl But it is not limited thereto.

Since the thermostabilized human serum albumin provided according to the method of the present invention is intended to be formulated and administered to humans as described above, its preparation method further includes a heat treatment step for blocking the contamination and / or the possibility of infection .

Preferably, the step may include heating the human serum albumin to 60 to 80 째 C, more preferably 65 to 75 째 C, and most preferably 70 째 C, but is not limited thereto. At this time, even when heated at the above temperature for 6 to 15 hours, preferably for 8 to 12 hours, more preferably for 10 hours, the α-Helix of the human serum albumin is maintained .

In addition, the method of the present invention may further comprise the step of concentrating the human serum albumin after the heat treatment to a concentration of 50 to 200 mg / ml. Specifically, this step corresponds to the step of formulating a low-concentration, heat-stabilized human serum albumin at a concentration of 1 to 40 mg / ml and concentrating it to an appropriate concentration for administration to an individual including a human.

The concentration may be performed by a generally used concentration method, for example, an ultrafiltration membrane having MWCO (Molecular Weight Cut Off) of 10 to 30 kD can be used to concentrate the solution to a proper concentration by centrifugation , But is not limited thereto.

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

In one embodiment of the present invention, human serum albumin at a concentration of 1 mg / ml was treated with oleic acid at a molar ratio of 1: 1 and heated at 60 to 80 ° C for 10 hours, 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 ° C and 70 ° C, but the stability was lowered at 80 ° C or higher (Examples 1 to 3, Figs.

Further, it was confirmed that stability was maintained even after heating oleic acid at a molar ratio of oleic acid to human serum albumin of 1 to 40 mg / ml at a molar ratio of 1: 1 and heating at 70 캜 for 10 hours (Experimental Example 4, Fig. 4) In addition, it was confirmed that the stability was maintained even after heating fatty acids of linoleic acid,? -Linolenic acid,? -Linolenic acid and palmitoleic acid and heating at 70 占 폚 for 10 hours.

This suggests that the low-concentration human serum albumin of 1 to 40 mg / ml is significantly increased in thermal stability by the addition of a fatty acid having 16 to 24 carbon atoms such as oleic acid at a temperature of 60 to 80 ° C.

In another aspect, the present invention provides a method of treating a human serum albumin comprising: (a) treating a human serum albumin with a fatty acid having from 16 to 24 carbon atoms or a pharmaceutically acceptable salt thereof; (b) adjusting the concentration of the human serum albumin to a concentration of 1 to 40 mg / ml. The present invention also provides a method for producing heat stabilized human serum albumin.

The fatty acids having 16 to 24 carbon atoms, or pharmaceutically acceptable salts thereof and human serum albumin are as described above.

In the manufacturing method of the present invention, the step (a) may include a step of stabilizing human serum albumin by treating a human serum albumin with a fatty acid selected from the group consisting of fatty acids having 16 to 24 carbon atoms or a pharmaceutically acceptable salt thereof, to be.

In step (b) of the present invention, the concentration of human serum albumin in the first step may be adjusted to a concentration of 1 to 40 mg / ml, so that a low concentration of human serum albumin In the second step. By controlling the concentration of the human serum albumin treated with the fatty acid having 16 to 24 carbon atoms or its pharmaceutically acceptable salt through the step (a) at a concentration of 1 to 40 mg / ml in this step, 10 The human serum albumin can be heat-treated stably for more than an hour.

The method of the present invention may further comprise the step of heating the heat stabilized human serum albumin to 60-80 占 폚. As described above, the heat stabilized human serum albumin according to the present invention corresponds to a heat treatment process for blocking the contamination and / or the possibility of infection for administration to human. Preferably, the heat treatment step performed to prevent contamination and / or infection may be performed after the step of treating the human serum albumin with a fatty acid having 16 to 24 carbon atoms.

In addition, the method of the present invention may further include the step of concentrating the human serum albumin after the heat treatment to a concentration of 50 to 200 mg / ml, as described above.

The heat-stabilized human serum albumin prepared by the above process may be administered to a human. In this case, the preferred method and formulations may be injections such as intravenous injection, subcutaneous injection, intradermal injection, intramuscular injection, But is not limited thereto. Injection may be carried out by using an aqueous solvent such as physiological saline solution or ring gel solution, a non-aqueous solvent such as vegetable oil, higher fatty acid ester (eg, oleic acid), alcohol (eg, ethanol, benzyl alcohol, propylene glycol, glycerin etc.) (For example, ascorbic acid, sodium hydrogen sulfite, sodium pyrophosphate, BHA, tocopherol, EDTA and the like), emulsifiers, buffers for pH control, preservatives for inhibiting microbial growth (For example, mercury nitrate, thimerosal, benzalkonium chloride, phenol, cresol, benzyl alcohol, etc.), and the like, but are not limited thereto.

By using the production method of the present invention, it is possible to produce human serum albumin in which stability is maintained even when heat treatment is performed at a high temperature of 60 DEG C or more for 10 hours or more. Therefore, it can be usefully used for the production of human serum albumin preparations (for example, injections, etc.) in which physiological activity is maintained without side effects.

1 shows the results obtained when 5 human serum albumin samples (Green Cross Albumin, A3782 albumin, A1653 albumin, Green Cross albumin + oleic acid (1: 1 molar ratio) and A3782 albumin + oleic acid (1: 1 molar ratio) And a circularly polarized light dichroism.
FIG. 2 shows the results obtained when 5 human serum albumin samples (Green Cross Albumin, A3782 albumin, A1653 albumin, Green Cross albumin + oleic acid (1: 1 molar ratio) and A3782 albumin + oleic acid (1: 1 molar ratio) And a circularly polarized light dichroism.
3 is a graph showing the results when 5 human serum albumin samples (Green Cross Albumin, A3782 albumin, A1653 albumin, Green Cross albumin + oleic acid (1: 1 molar ratio) and A3782 albumin + oleic acid (1: 1 molar ratio) And a circularly polarized light dichroism.
FIG. 4 shows the result of measuring circular dichroism dichroism when 10 human serum albumin samples (1 to 45 mg / ml A3782 albumin + oleic acid (1: 1 molar ratio)) with different concentrations were heated at 70 ° C for 10 hours Graph.
FIG. 5 is a graph showing the results of the measurement of 5 human serum albumin samples (A3782 albumin + oleic acid (1: 1 molar ratio), A3782 albumin + linoleic acid (1: 1 molar ratio), A3782 albumin +? - linolenic acid (1: 1 molar ratio), A3782 albumin + palmitoleic acid (1: 1 molar ratio)) was heated at 70 DEG C for 10 hours.

Hereinafter, the present invention will be described more specifically in the following examples. However, these examples are provided only to aid understanding of the present invention, and the present invention is not limited thereto.

Example  : Preparation of human serum albumin sample

A3782 albumin (Sigma) and A1653 albumin (Sigma) were each diluted with 20 mM potassium phosphate buffer (pH 7.5) to 1 mg / ml. Then, oleic acid was added to each of green blood albumin and A3782 albumin such that the molar ratio of oleic acid to albumin was 1: 1, and then reacted overnight at 4 ° C to prepare 5 human serum albumin samples; (1: 1 molar ratio); (v) Green Cross Albumin + oleic acid (1: 1 molar ratio); (iii) Green Cross (room temperature), (ii) A1653 albumin, (iii) A3782 albumin, (iv) A3782 albumin and oleic acid.

Experimental Example  1: of human serum albumin Thermal stability  exam

(1) albumin Circular polarization dichroism ( circular dichroism ) How to measure

Circular dichroism can predict the composition ratio of the secondary structure of the protein and can measure the structural change of the thermally denatured protein. The untreated native Green Cross albumin and the five thermally denatured albumin samples were diluted with 20 mM potassium phosphate buffer (pH 7.5) to 0.25 mg / ml.

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

(2) the ratio of human serum albumin Thermal stability  exam

5 human serum albumin samples (Green Cross Albumin, A3782 albumin, A1653 albumin, Green Cross albumin + oleic acid (1: 1 molar ratio) and A3782 albumin + oleic acid (1: 1 molar ratio)) prepared in the above example were heated at 60 DEG C for 10 hours Respectively.

Thereafter, as a result of circular dichroism measurement by the above method (1), the composition ratio of the secondary structure of the albumin sample was as shown in Table 1 below.

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 1, the ratio of the α-Helix of thermally denatured specimens was substantially reduced and the number of unordered structures was increased compared to the untreated Green Cross (normal temperature) Respectively.

However, the ratio of Green Cross albumin + oleic acid (1: 1) sample was similar to that of Green Cross (room temperature) specimen, and it was found that it was stable even after heating at 60 ° C for 10 hours. In addition, the A3782 albumin + oleic acid (1: 1) sample also showed higher thermal stability than the A3782 albumin after the thermal denaturation because the ratio of α-Helix was higher. This suggests that when oleic acid is reacted with albumin, thermal stability is increased.

Experimental Example  2: the ratio of human serum albumin at < RTI ID = 0.0 > 70 C & Thermal stability  exam

Five human serum albumin samples (Green Cross Albumin, A3782 albumin, A1653 albumin, Green Cross albumin + oleic acid (1: 1 molar ratio) and A3782 albumin + oleic acid (1: 1 molar ratio)) prepared in the above example were heated at 70 DEG C for 10 hours Respectively.

Thereafter, as a result of circular dichroism measurement by the above method (1), the composition ratio of the secondary structure of the albumin sample was as shown in Table 2 below.

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 a result of the heat treatment as described above, the proportion of the α-Helix of the thermally denatured specimens was reduced and the unordered structure was increased compared to the untreated Green Cross (room temperature) specimen .

However, the proportion of α-Helix in the A782 albumin + oleic acid (1: 1 molar ratio) specimen after the reaction with oleic acid and then heat-treated Green Cross-albumin + oleic acid (1: 1 molar ratio) , And it was confirmed that even when heated at 70 ° C for 10 hours, it was very stable compared to albumin not reacted with oleic acid.

Experimental Example  3: Preparation of human serum albumin at < RTI ID = 0.0 > 80 C & Thermal stability  exam

5 human serum albumin samples (Green Cross Albumin, A3782 albumin, A1653 albumin, Green Cross albumin + oleic acid (1: 1 molar ratio) and A3782 albumin + oleic acid (1: 1 molar ratio)) prepared in the above example were heated at 80 DEG C for 10 hours Respectively.

Thereafter, as a result of circular dichroism measurement by the method (1), the composition ratio of the secondary structure of the albumin sample was as shown in Table 3 below.

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 a result of the heat treatment, the proportion of α-Helix of all thermally denatured specimens was reduced and the number of unordered structures increased compared to that of the green cross (room temperature) without heat denaturation.

However, unlike at 60 ° C and 70 ° C, the proportion of α-Helix in both A3782 albumin and oleic acid (1: 1 molar ratio) decreased and the ratio of unordered ) Structure.

Experimental Example  4: Thermal Stability  Test the concentration range of albumin to be maintained

A3782 Albumin (Sigma) was dissolved in 20 mM potassium phosphate (Sigma) to a concentration of 1 mg / ml, 5 mg / ml, 10 mg / ml, 15 mg / ml, 20 mg / ml, 25 mg / ml, 30 mg / ml, 35 mg / and diluted with pH 7.5 buffer. Then, oleic acid was added to A3782 albumin so that the mole ratio of the oleic acid to the albumin became 1: 1, followed by reaction at 4 ° C overnight.

(1: 1 mole ratio), 5 mg / ml A3782 albumin + oleic acid (1: 1 mole ratio), 10 mg / ml A3782 albumin + oleic acid (1: 1 mole ratio), 15 mg / ml A3782 albumin + Oleic acid (1: 1 mole ratio), 20 mg / ml A3782 albumin + oleic acid (1: 1 mole ratio), 25 mg / ml A3782 albumin + oleic acid (1: 1 mole ratio), 30 mg / ml A3782 albumin + 10 kinds of specimens such as 35 mg / ml A3782 albumin + oleic acid (1: 1 molar ratio), 40 mg / ml A3782 albumin + oleic acid (1: 1 molar ratio) and 45 mg / ml A3782 albumin + oleic acid For 10 hours.

The circular dichroism of albumin was measured according to the method of Experimental Example 1- (1). As a result, a sample of 1 mg / ml A3782 albumin + oleic acid (1: 1 molar ratio) The ratio of the sample to the α-helical structure was similar (Table 2 and FIG. 2). 1 to 40 mg / ml of A3782 albumin + oleic acid (1: 1 molar ratio) in a 1: 45 mg / ml A3782 albumin + oleic acid (1: 1 molar ratio) It was confirmed that the composition ratio of the protein secondary structure was not largely changed due to the similar spectrum of albumin + oleic acid (1: 1 molar ratio) (FIG. 4).

On the other hand, the 45 mg / ml A3782 albumin + oleic acid (1: 1) specimens significantly decreased the signal of the circular dichroism, and the ratio of α-Helix was decreased and the unordered structure was increased. Therefore, it was confirmed that A3782 albumin + oleic acid (1: 1 molar ratio) samples retained high thermal stability within the range of 1 to 40 mg / ml (FIG. 4).

Experimental Example  5: Carbon number  Of albumin due to the addition of 16 to 24 unsaturated fatty acids Enlightenment Qualitative evaluation

A3782 albumin (Sigma) was diluted with 20 mM potassium phosphate pH 7.5 buffer to 1 mg / ml. Then, oleic acid, linoleic acid,? -Linolenic acid,? -Linolenic acid, and palmitoleic acid were added to A3782 albumin so that the ratio of mole ratio to albumin was 1: 1.

A3782 albumin + alpha-linolenic acid (1: 1 molar ratio), A3782 albumin + gamma-linolenic acid (1: 1 molar ratio), A3782 albumin + oleic acid (1: 1 molar ratio), A3782 albumin + linoleic acid + Palmitoleic acid (1: 1 molar ratio) were heated at 70 占 폚 for 10 hours.

The circular dichroism of albumin was measured according to the method of Experimental Example 1- (1). As a result, a sample of A3782 albumin + oleic acid (1: 1 molar ratio) - The ratio of the helical structure (a-Helix) was similar (Table 2 and Fig. 2). In a circular dichroism dichroism graph of an albumin sample added with thermally denatured 16 to 24 unsaturated fatty acids, a sample to which an unsaturated fatty acid having a carbon number of 16 to 24 except for oleic acid was added generally had a sample containing oleic acid and a circularly polarized dichroism spectrum Similarly, it was confirmed that there was no significant change in the secondary structure of the protein (Fig. 5).

These results suggest that a fatty acid having 16 to 24 carbon atoms in addition to oleic acid may be used in the method of the present invention to obtain the same or corresponding effect.

From the above description, it will be understood by those skilled in the art that the present invention may be embodied in other specific forms without departing from the spirit or essential characteristics thereof. In this regard, it should be understood that the above-described examples are illustrative in all aspects and not restrictive. The scope of the present invention should be construed as being included in the scope of the present invention, rather than the above detailed description, as well as all changes or modifications derived from the meaning and scope of the appended claims and their equivalents.

<110> Ewha University - Industry Collaboration Foundation <120> Method for preparing thermal-stabilized human serum albumin <130> PA130370 / KR <160> 1 <170> Kopatentin 2.0 <210> 1 <211> 585 <212> PRT <213> Homo sapiens <400> 1 Asp Ala His Lys Ser Glu Val Ala His Arg Phe Lys Asp Leu Gly Glu   1 5 10 15 Glu Asn Phe Lys Ala Leu Val Leu Ile Ala Phe Ala Gln Tyr Leu Gln              20 25 30 Gln Cys Pro Phe Glu Asp His Val Lys Leu Val Asn Glu Val Thr Glu          35 40 45 Phe Ala Lys Thr Cys Val Ala Asp Glu Ser Ala Glu Asn Cys Asp Lys      50 55 60 Ser Leu His Thr Leu Phe Gly Asp Lys Leu Cys Thr Val Ala Thr Leu  65 70 75 80 Arg Glu Thr Tyr Gly Glu Met Ala Asp Cys Cys Ala Lys Gln Glu Pro                  85 90 95 Glu Arg Asn Glu Cys Phe Leu Gln His Lys Asp Asp Asn Pro Asn Leu             100 105 110 Pro Arg Leu Val Arg Pro Glu Val Asp Val Met Cys Thr Ala Phe His         115 120 125 Asp Asn Glu Glu Thr Phe Leu Lys Lys Tyr Leu Tyr Glu Ile Ala Arg     130 135 140 Arg His Pro Tyr Phe Tyr Ala Pro Glu Leu Leu Phe Phe Ala Lys Arg 145 150 155 160 Tyr Lys Ala Ala Phe Thr Glu Cys Cys Gln Ala Ala Asp Lys Ala Ala                 165 170 175 Cys Leu Leu Pro Lys Leu Asp Glu Leu Arg Asp Glu Gly Lys Ala Ser             180 185 190 Ser Ala Lys Gln Arg Leu Lys Cys Ala Ser Leu Gln Lys Phe Gly Glu         195 200 205 Arg Ala Phe Lys Ala Trp Ala Val Ala Arg Leu Ser Gln Arg Phe Pro     210 215 220 Lys Ala Glu Phe Ala Glu Val Ser Lys Leu Val Thr Asp Leu Thr Lys 225 230 235 240 Val His Thr Glu Cys Cys His Gly Asp Leu Leu Glu Cys Ala Asp Asp                 245 250 255 Arg Ala Asp Leu Ala Lys Tyr Ile Cys Glu Asn Gln Asp Ser Ile Ser             260 265 270 Ser Lys Leu Lys Glu Cys Cys Glu Lys Pro Leu Leu Glu Lys Ser His         275 280 285 Cys Ile Ala Glu Val Glu Asn Asp Glu Met Pro Ala Asp Leu Pro Ser     290 295 300 Leu Ala Ala Asp Phe Val Glu Ser Lys Asp Val Cys Lys Asn Tyr Ala 305 310 315 320 Glu Ala Lys Asp Val Phe Leu Gly Met Phe Leu Tyr Glu Tyr Ala Arg                 325 330 335 Arg His Pro Asp Tyr Ser Val Val Leu Leu Leu Arg Leu Ala Lys Thr             340 345 350 Tyr Glu Thr Thr Leu Glu Lys Cys Cys Ala Ala Ala Asp Pro His Glu         355 360 365 Cys Tyr Ala Lys Val Phe Asp Glu Phe Lys Pro Leu Val Glu Glu Pro     370 375 380 Gln Asn Leu Ile Lys Gln Asn Cys Glu Leu Phe Lys Gln Leu Gly Glu 385 390 395 400 Tyr Lys Phe Gln Asn Ala Leu Leu Val Arg Tyr Thr Lys Lys Val Pro                 405 410 415 Gln Val Ser Thr Pro Thr Leu Val Glu Val Ser Arg Asn Leu Gly Lys             420 425 430 Val Gly Ser Lys Cys Cys Lys His Pro Glu Ala Lys Arg Met Pro Cys         435 440 445 Ala Glu Asp Tyr Leu Ser Val Val Leu Asn Gln Leu Cys Val Leu His     450 455 460 Glu Lys Thr Pro Val Ser Asp Arg Val Thr Lys Cys Cys Thr Glu Ser 465 470 475 480 Leu Val Asn Arg Arg Pro Cys Phe Ser Ala Leu Glu Val Asp Glu Thr                 485 490 495 Tyr Val Pro Lys Glu Phe Asn Ala Glu Thr Phe Thr Phe His Ala Asp             500 505 510 Ile Cys Thr Leu Ser Glu Lys Glu Arg Gln Ile Lys Lys Gln Thr Ala         515 520 525 Leu Val Glu Leu Val Lys His Lys Pro Lys Ala Thr Lys Glu Gln Leu     530 535 540 Lys Ala Val Met Asp Asp Phe Ala Ala Phe Val Glu Lys Cys Cys Lys 545 550 555 560 Ala Asp Asp Lys Glu Thr Cys Phe Ala Glu Glu Gly Lys Lys Leu Val                 565 570 575 Ala Ala Ser Gln Ala Ala Leu Gly Leu             580 585

Claims (11)

One or more fatty acids selected from the group consisting of palmitoleic acid, oleic acid, linoleic acid and linolenic acid, or a pharmaceutically acceptable salt thereof, in an amount of 1 to 40 mg / ml To a human serum albumin at a concentration of about 1: 1,
Characterized in that the α-helical structure (α-Helix) is maintained at 80% or more even when the thermally stabilized human serum albumin is heat-treated at a temperature of 60 to 80 ° C.
delete delete The process according to claim 1, wherein the pharmaceutically acceptable salt of the fatty acid is an alkali metal or alkaline earth metal salt.
The method according to claim 1, wherein the fatty acid or a pharmaceutically acceptable salt thereof is added in a molar ratio of 1: 0.5 to 1: 2 to human serum albumin.
The method according to claim 1, wherein the human serum albumin is human plasma-derived or recombinant human serum albumin.
The method according to claim 1, wherein the method further comprises heating the human serum albumin to 60 to 80 캜.
The method according to claim 1, wherein the method further comprises concentrating the human serum albumin to a concentration of 50 to 200 mg / ml.
(a) contacting the human serum albumin with one or more fatty acids selected from the group consisting of palmitoleic acid, oleic acid, linoleic acid and linolenic acid, or a pharmaceutically acceptable salt thereof, Processing;
(b) adjusting the concentration of the human serum albumin to a concentration of 1 to 40 mg / ml,
Characterized in that the α-helical structure (α-Helix) is maintained at 80% or more even when the thermally stabilized human serum albumin is heat-treated at a temperature of 60 to 80 ° C.
10. The method of claim 9, further comprising: (c) heating the human serum albumin to 60-80 &lt; 0 &gt; C.
11. The method of claim 10, further comprising: (d) concentrating the human serum albumin to a concentration of 50-200 mg / ml.

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