KR20080062328A - Method of purifying apoa-i and method of producing reconstituted high density lipoprotein by using the purified apoa-i - Google Patents

Abstract

A method for purifying ApoA-I(apolipoprotein A-I) is provided to mass-purify economically the ApoA-I with high purity. A method for producing reconstituted high density lipoprotein(rHDL) using the purified ApoA-I is provided to mass-produce the rHDL with high purity and excellent virus stability, thereby the produced rHDL being usefully used as a composition for preventing or treating cardiovascular diseases, sepsis or anti-inflammation. A method for purifying ApoA-I comprises the steps of: (a) extracting proteins including the ApoA-I from a material including the ApoA-I; (b) treating the proteins with a precipitating agent to remove protein impurities with high molecular weight therefrom; (c) treating a solution obtained from the step(b) with a precipitating agent to precipitate the proteins including the ApoA-I; (d) after dissolving the precipitated proteins in a buffer solution, treating it with ethanol to remove protein impurities with high molecular weight therefrom; (e) adjusting a pH of an ethanol-treated supernatant to collect the precipitated material; (f) after dissolving the precipitated material in a buffer solution or a chaotropic solution, heat-treating it to inactivate virus; (g) subjecting a product obtained from the step(f) to chromatography to isolate the ApoA-I therefrom; and (h) nano-filtering the isolated ApoA-I to remove virus therefrom. A method for producing rHDL comprises the steps of: (a) adding a lipid and a surfactant to the ApoA-I solution purified by the method above and agitating it enough at low temperature; ad (b) dialyzing a product obtained from the step(a) using an ultrafiltration membrane. Further, a concentration of the ApoA-I is 1 to 10 mg/ml, and a molar ratio of the ApoA-I, the lipid and the surfactant is 1:100~200:150~250.

Description

Method for Purification of AA-I and Production of Reconstituted HDL Using Purified AA-I {METHOD OF PURIFYING ApoA-I AND METHOD OF PRODUCING RECONSTITUTED HIGH DENSITY LIPOPROTEIN BY USING THE PURIFIED ApoA-I}

1 is a process diagram illustrating a process of purifying ApoA-I and rebuilding high density lipoprotein (HDL) using purified ApoA-I,

Figure 2 is a SDS-PAGE results confirming the molecular weight and purity of ApoA-I purified in each step of Example 1,

3A is an electron micrograph of reconstituted HDL (rHDL, reconstituted HDL) of soybean phosphatidyl choline (SoyPC) in Example 2,

Figure 3b is HDL (rHDL) reconstructed with POPC (1-palmitoyl 2-oleoyl phosphatidyl choline) in Example 3.

The present invention relates to an effective method for purifying apolipoprotein A-I (ApoA-I) and a method for producing reconstituted high density lipoprotein (rHDL) using purified ApoA-I.

Atherosclerosis is an inflammation-related disease in which lipid-rich plaque accumulates on the inner wall of the aorta, including myocardial infarction, stroke, and peripheral vascular disease (Hansson, GK, ( 2005) N. Engl. J. Med. 352: 1685-1695). These lipid lesions are caused by low density lipoprotein (LDL), and currently used lipid-modifying drugs may lower the risk of cardiovascular disease. However, there are limitations to the treatment of cardiovascular diseases that simply lower LDL-cholesterol levels.

According to past clinical and epidemiological reports, high density lipoprotein (HDL) -cholesterol concentrations and the risk of cardiovascular disease show contradictory relationships (Miller, GJ and Miller, NE, (1975) Lancet 1: 16-25; Gordin, T., et al., (1977) Am. J. Med. 62: 707-714; and Castelli, WP et al., (1986) JAMA 256: 2835-2838). From this point of view, since HDL-cholesterol levels, rather than LDL-cholesterol levels, are important factors for cardiovascular disease, many pharmaceutical companies are focusing on developing drugs that raise HDL-cholesterol levels. However, this is only a precaution and there are still many questions about whether HDL-cholesterol levels actually affect the incidence of cardiovascular disease.

HDL and its major constituent, ApoA-I, are important regulators of "reverse cholesterol transport" (RCT), which extracts free cholesterol from peripheral cells and delivers it to the liver (Brewer). , HB, (2004) N. Engl. J. Med. 350: 1491-1494). HDL also protects LDL from oxidation (see Mackness, MI et al., (1993) Atherosclerosis 104: 129-135), anti-inflammatory, anti-thrombotic and Has pro-fibrolytic properties (Shah, PK et al., (2001) Circulation 104: 2376-2383; Kuvin, JT and Karas, RH, (2003) Curr. Opin. Cardiol. 18: 295-300; and Ansell, BJ et al., (2004) Rev. Endocr. Metab. Disord. 5: 351-358).

In addition, ApoA-I is derived from a precursor consisting of 267 amino acids and exists in the blood as 243 amino acids, but many variants have been reported (Brewer et al., Biochem. Biophys. Res. Commun. (1978) 80: 623 -630 and Frank, PG and Marcel, YL, (2000) J. Lipid Res. 41: 853-872). The structural feature of ApoA-I is that the 11 or 22 amino acids make up a repeating unit that has an amphipathic helical conformation (Segrest et al., FEBS Lett. (1974) 38: 247-253) . This imparts ApoA-I lipid binding capacity or lecithin cholesterol acyl transferase (LCAT) activity.

Although the ability of ApoA-I or HDL to reduce atherosclerosis has been confirmed in experimental animals, it is necessary to secure mass production and stability before developing it as a therapeutic agent. The ultracentrifugation method has been classically used to separate HDL from plasma. However, this method is limited to the small amount of separation at the laboratory level. Therefore, HDL can be obtained by separating ApoA-I from plasma and mixing externally obtained lipids. There has been a way to rebuild the system.

The most common method for purifying ApoA-I from plasma is to separate HDL using an ultracentrifuge and then to separate ApoA-I from HDL using an organic solvent extraction method. However, this method is uneconomical and not suitable for mass production.

In addition, methods for producing ApoA-I from plasma include chromatography methods (see Ross SE et al., (1985) Anal. Biochem. 149: 166-168) and gel filtration HPLC methods (Wehr, CT). et al., (1982) Anal. Biochem. 125: 386-394).

For example, WO 93/12143 discloses the separation of ApoA-IM (Milano type of ApoA-I) from plasma by affinity chromatography and gelation of ApoA-IM produced using recombinant DNA technology. Techniques for separating by filtration chromatography have been presented, and US Pat. Nos. 6,090,921 and 6,423,830 describe a method for purifying ApoA-I from plasma using anion-exchange chromatography.

Meanwhile, a method for separating ApoA-I using a lower aliphatic alcohol or an organic solvent (see US Pat. No. 5,089,602) and an aqueous two-phase using a salt and a surfactant The method for separating ApoA-I (see US Pat. Nos. 6,767,994 and 6,107,467) was also studied.

In addition, European Patent Publication No. 0329605 purifies ApoA-I from a fraction of plasma containing ApoA-I, and then precipitates B and IV in cold ethanol fractionation process can be an initial material capable of producing ApoA-I. In order to precipitate impurities, alcohol concentration conditions and an organic solvent were used, and guanidine-HCl was removed by gel filtration.

Methods of reconstitution of lipoproteins using ApoA-I, A-II, A-IV, ApoC, ApoE have already been known in detail (see A. Jonas, (1986) 128: 553-582). In this case, phosphatidylcholine (PC) has been mainly used for lipids. A general reconstruction method is to evaporate lipids dissolved in organic solvents under nitrogen gas, add lipoproteins and solvents, stir, and then dialysate to obtain reconstructed material, which is not suitable for mass production. A suitable reconstruction method for the production process is a method of mixing a protein solution with a pre-prepared lipid-solvent solution without using an organic solvent, stirring and dialysis to obtain a desired substance, as described in US Pat. No. 5,652,339. Meanwhile, US Patent No. 6,287,590 proposes a method for producing reconstituted protein / lipid compound by lyophilization without using a solvent.

The method described above describes a part of the steps in the production of rHDL as a therapeutic agent for cardiovascular disease, for example, the separation and reconstitution of ApoA-I, and substantially the high purity of the product and It does not meet the requirements of plasma-derived medical products that require viral safety. In addition, it does not take into account the scale-up and cost issues necessary to meet the expected production of rHDL.

Accordingly, it is an object of the present invention to provide a method for effectively purifying apolipoprotein A-I (ApoA-I).

Another object of the present invention is to provide a method for efficiently mass-producing reconstituted HDL (rHDL) useful for treating cardiovascular disease, sepsis or anti-inflammatory using ApoA-I purified according to the above method.

According to the above object, the present invention is obtained by extracting the protein containing ApoA-I from the material containing ApoA-I (extracting step), and by removing the high-molecular weight protein impurities by treating the precipitant (first precipitation step) Secondary treatment of the precipitant in the solution to precipitate the protein containing ApoA-I (secondary precipitation step), the precipitate was dissolved in a buffer solution and then treated with ethanol to remove high molecular weight protein impurities again (ethanol precipitation Step), to recover the precipitate by adjusting the pH of the ethanol-treated supernatant (pH precipitation step), the precipitate is dissolved in a buffer or chaotropic solution and heat treated to inactivate the virus (virus inactivation step), Separation of ApoA-I by chromatography (chromatography step) and nanofiltration to remove virus (nanofiltration step). Provide a purification method.

According to the above another object, the present invention reconstitution comprising the step of adding a lipid and a surfactant to the purified ApoA-I solution at the same time and sufficiently stirred at low temperature, and then using an ultrafiltration membrane for dialysis. Provides a method of producing HDL.

Hereinafter, the present invention will be described in detail.

ApoA-I purification method of the present invention,

1) A solution containing ApoA-I by dissolving a material comprising ApoA-I, preferably from plasma or dissolving the recombinant ApoA-I culture with urea, guanidine-HCl, alcohol or a mixture thereof, and then centrifuging or filtering. An extraction step of obtaining;

2) a first precipitation step of precipitating a high molecular weight protein impurity by adding and stirring a precipitant to the solution obtained in step 1), followed by centrifugation or filtration to recover a solution containing ApoA-I;

3) a second precipitation step of precipitating the protein comprising ApoA-I by adding a precipitant to the solution obtained in step 2), and then recovering the precipitate by centrifugation or filtration;

4) an ethanol precipitation step of dissolving the precipitate obtained in step 3) in a buffer of pH 7 or higher, and then adding ethanol to precipitate impurities, and then centrifuging or filtering to recover a solution containing ApoA-I;

5) pH precipitation step of adjusting the pH of the solution obtained in step 4) to 5 to 7 to precipitate the protein containing ApoA-I, and then recovering the precipitate by centrifugation or filtration;

6) virus inactivation step of dissolving the precipitate obtained in step 5) in a buffer or a chaotropic solution of pH 7 or more, and then heat-treated at 60 ℃ for 10 hours;

7) chromatography of the heat-treated solution obtained in step 6) using anion exchange resin; And

8) characterized in that it comprises the step of nanofiltration the eluate obtained in step 7).

Looking at each step of the ApoA-I purification method according to the invention in detail as follows (see Fig. 1):

1) Extraction step

The substance for separating ApoA-I in the present invention may preferably be derived from plasma or may be a recombinant ApoA-I culture, more preferably any fraction isolated from plasma as long as it contains ApoA-I, most preferably Cohn fraction IV obtained by treating plasma with cold ethanol fractionation can be used.

Cohn fraction IV obtained as described above precipitates not only lipoproteins but also protein impurities such as filter aids (eg, supercel or celite) and albumin. Therefore, in this step, urea, guanidine-HCl, alcohol, for example, ethanol, or a mixture thereof is used as the extract to completely extract the filter aid contained in Cohn Fraction IV. Preferably, the extraction is performed at about 4 ° C. to 20 ° C. for 1 to 3 hours, but is not limited thereto. The concentration of the urea is 4 M or more, preferably 6 to 8 M.

Once extraction is complete, the mixture is centrifuged or filtered to obtain a solution comprising ApoA-I.

In addition, in this step, a washing process may be introduced before the extraction process to remove protein impurities from Cohn fraction IV. NaCl, an organic acid such as citric acid, a mineral such as phosphate may be used as the washing liquid, and washing is preferably performed at 3 ° C. to 20 ° C. for 1 to 3 hours, but is not limited thereto.

In this step, the washing solution is added to Cohn Fraction IV to adjust the pH to 4 to 6, and then suspended using a stirrer. The suspension is filtered using a filter press to obtain a precipitate, or centrifuged. The supernatant can be discarded to obtain various protein precipitates including ApoA-I. At this time, ApoA-I is hardly removed and is present in more than 90% of the precipitate.

2) 1st precipitation step

In this step, to remove a large amount of high molecular weight proteins and lipids in the solution obtained in step 1), polyethylene glycol (PEG, polyethylene glycol) or ammonium sulfate (preferably PEG) may be used as a precipitant, PEG 4000, PEG 6000, PEG 8000 or PEG 20000, preferably PEG 4000, is added to the solution obtained in step 1 at a concentration of about 5 to 20% (W / V), and then 4 ° C. or higher, preferably Is stirred at 18-25 ° C. for about 1-5 hours, and centrifuged or filtered to precipitate large amounts of high molecular weight proteins and lipids to obtain a solution comprising ApoA-I.

3) Second precipitation step

In this step, in order to precipitate the protein comprising ApoA-I in the solution obtained in step 2), the solution obtained in the first precipitation step is added with a precipitant, preferably PEG, about 5 to 40% (W / V), Preferably it is added at a concentration of 10 to 30% (W / V) and treated under the same conditions as in step 2 to precipitate a protein comprising ApoA-I.

In this step, the overall reaction volume can be reduced and the yield can be increased to be suitable for mass production compared with the conventional method of performing only the primary precipitation, and known methods used for protein precipitation, for example salt and metal ions, can be used instead of precipitant addition. The protein containing ApoA-I may be precipitated by adjusting the concentration or pH of the same.

4) Ethanol Precipitation Step

In this step, to dissolve the secondary precipitate, a buffer of pH 7 or higher, preferably pH 7 to 9, such as Tris buffer, phosphate buffer, or the like is added to the precipitate. At this time, the buffer is preferably used so that the protein concentration of the completely dissolved solution is 5 to 25 mg / ㎖.

In this step also 50-75%, preferably 60-75% ethanol is added to the solution and allowed to stand at 4-25 ° C. for 5 to 24 hours to precipitate impurities to obtain a supernatant.

At this time, in order to improve the sedimentation ability to improve the shape of the precipitate, after the addition of ethanol, salts or metal ions may be further added at a concentration of 2 to 30 mM, and most preferably 2 to 20 to 60 to 75% of ethanol. By adding a salt or a metal ion of mM to adjust the ionic strength of the solution (2 to 20 mS / cm) can obtain an excellent purification effect.

5) pH precipitation step

In this step, in order to recover ApoA-I from the ethanol supernatant, precipitation is induced by adjusting the pH of the solution close to the isoelectric point of ApoA-I. At this time, a suitable pH is 5 to 7, preferably 5 to 6.5, the precipitate is recovered by centrifugation or filtration after stirring.

Even at this stage, the overall reaction volume can be reduced to be suitable for mass production, and the recovered precipitate is suitable for long-term storage or distribution.

6) Virus Inactivation Step

In order to increase the viral safety of purified ApoA-I, the pH precipitate may be treated with a buffer of pH 7 or higher, e.g., Tris buffer, phosphate buffer or chaotropic solution (e.g. 4-8 M urea or 4-8 M guanidine-HCl solution), followed by heat treatment at 60 ° C. for at least 10 hours to inactivate viruses such as AIDS and hepatitis B virus contained in the pH precipitate, and at the same time ApoA-I (see RA Gangani et al., Biochemistry (2005) 44: 2759-2769), which existed as monomers and dimers due to self association properties, is modified with monomers do.

7) Chromatography Step

In the protein purification method, lipids are removed by precipitating proteins using an organic solvent such as ethanol, diethyl ether, hexane, isopropanol, and then filled with nitrogen to remove the organic solvent, and then dissolved by guanidine-HCl. Although dialysis is used, this method is possible at the laboratory level and is not suitable for mass production.

Therefore, in this step, chromatography is performed using an anion exchange resin to remove lipids, protein impurities and remaining alcohol from the heat treated solution. Preferably, anions such as agarose bound with quaternary amines (eg Q-Sepharose) and diethylaminoethyl (DEAE) bound agarose (DEAE-Sepharose) Using an exchange resin, the buffer is preferably Tris-HCl, Tris, triethanolamine, sodium phosphate or glycineamide, in which the lipids and ureas do not bind to the resin and exit as nonadsorbents and ApoA-I is dissolved in salts. Eluted, the purified ApoA-I solution is at least 90% pure.

8) Nanofiltration Step

In the present invention, nanofiltration is performed to remove viruses from the ApoA-I solution eluted in the above step.

In addition, the present invention is a lipid-surfactant solution prepared by dissolving a surfactant in a buffer, and then adding a lipid to the purified ApoA-I and sufficiently stirred at low temperature, followed by dialysis using an ultrafiltration membrane In the existing method for producing reconstituted HDL comprising the step of adding a lipid and a surfactant to the purified ApoA-I solution at the same time, the process is simple and economical production method of reconstituted HDL to provide.

At this time, the protein concentration of the ApoA-I solution is 1 to 10 mg / ㎖, the molar ratio (molar ratio) of protein, lipid and surfactant is 1: 100 to 200: 150 to 250, stirring conditions are 2 to 10 ℃ In 4 to 24 hours.

In addition, the size of the ultrafiltration membrane is 1,000 to 100,000 Da, the pH of the buffer used during dialysis is preferably 6 to 10, and may further include a small amount of carbohydrates such as 1% (W / V) sucrose. .

As the lipid, phospholipids or synthetic phospholipids purified from soybeans or eggs may be used, and as the surfactant, cholate, deoxycholate or urosodeoxycholate may be used. A bile acid or bile salt containing ursodeoxycholate can be used.

In addition, in the present invention, 5 to 15% (W / V) of conventional disaccharides (e.g. sucrose, maltose, lactose, etc.) or 1 to 10% (W / Conventional monosaccharides of V) (eg glucose, fructose, galactose, ribose, deoxyribose, etc.), preferably sucrose, can be used, preferably lyophilized for long term storage.

rHDL binds to lipids and fatty substances in an individual and has characteristics that affect their transport and activity. Thus, rHDL is useful for the prevention and treatment of various diseases associated with lipids and fatty substances.

Therefore, the rHDL produced according to the present invention can be usefully used for the prevention or treatment of cardiovascular diseases, sepsis or anti-inflammatory, including hypertension, angina pectoris, myocardial infarction, stroke and cerebrovascular diseases, etc. It can also be used as a carrier or extended release material.

When rHDL produced according to the present invention is used for the prevention or treatment of cardiovascular disease, sepsis or anti-inflammatory, etc., the pharmaceutical composition comprising the rHDL as an active ingredient or in the body of a patient according to a conventional method in the pharmaceutical field It may be administered after being formulated into a dosage unit formulation suitable for administration.

The composition or formulation can be administered via several routes including oral, transdermal, subcutaneous, intravenous or intramuscular, preferably through arteries, intraperitoneal or spinal cavity, for which pharmaceutically acceptable addition to rHDL is used as the active ingredient. Possible carriers may additionally be included, for example water soluble carriers, excipients or, if desired, other additives. The formulation may be in the form of tablets, pills, powders, crudes, elixirs, suspensions, emulsions, solutions, syrups, aerosols, soft or hard gelatin capsules, sterile injectables, sterile powders and the like.

The daily dose of rHDL may be administered 15 to 80 mg / kg body weight, preferably 40 to 80 mg / kg body weight once or divided into several times. However, it is to be understood that the actual dosage of the active ingredient should be determined in light of several relevant factors such as the disease to be treated, the severity of the disease, the route of administration, the patient's weight, age and gender, and therefore the dosage may be determined in some way. It does not limit the scope of the invention.

Hereinafter, the present invention will be described in more detail with reference to the following examples. However, the following examples are only for illustrating the present invention, and the scope of the present invention is not limited thereto.

Example 1

<1-1> Purification of ApoA-I

To the 150 kg Cohn Fraction IV paste (Green Cross) was added 400 L of wash solution (70 mM citrate, 80 mM sodium phosphate, pH 4.0) to adjust the pH to 4.1 to 4.2. Suspended for hours. A precipitate was obtained from the suspension by using a filter press (Korea Filter, Inc.). 600 L of 8 M urea was added to the precipitate and suspended at room temperature for 2 hours, and then the filtrate was recovered using a filter (extraction step).

119 kg of PEG 4000 (Sanyo, Japan) was added to the filtrate to give a PEG concentration of 17% (W / V), followed by stirring at 20 ° C. for 2 hours, followed by filtration to recover the filtrate (first precipitation). step).

138 kg of 20% (W / V) PEG 4000 was added to the filtrate to a final PEG concentration of 37% (W / V), followed by stirring for 2 hours and filtered to recover the precipitate (secondary precipitation). step).

300 L of buffer (30 mM Tris-HCl, pH8.0) was added to the PEG precipitate and completely dissolved by stirring. Then, 600 L of 66% ethanol was added and allowed to stand overnight at 18 ° C. The precipitate was removed (ethanol precipitation step).

The pH of the ethanol supernatant obtained above was adjusted to 6.0 using 1 M HCl, and the precipitate containing ApoA-I was collected by a filter and stored in the refrigerator (pH precipitation step).

At this time, the reaction volume of each step, the total protein concentration and content, the concentration and content of the purified ApoA-I, and the yield are shown in Table 1 below, the molecular weight and purity of the purified ApoA-I SDS-PAGE As a result, ApoA-I of about 29 KDa was obtained with a purity of 95% (FIG. 2).

As shown in Table 1 and Figure 2, using the ApoA-I purification method of the present invention, ApoA-I with improved virus safety can be obtained in high purity and high yield.

<1-2> Removal of Virus

350 g of the pH precipitate obtained in <1-1> were dissolved in 100 L of the same buffer or 8M urea as used in the ethanol precipitation step, and then heated at 60 ° C. for 16 hours (virus inactivation step).

The heat treated ApoA-I solution was adsorbed onto a DEAE-Sepharose column equilibrated with the buffer (GE-Health care, USA; 30 × 35 cm) to remove unadsorbed traces of lipids and urea, followed by ApoA with 250 mM NaCl. -I eluted (chromatography step).

Viresolve ^® using the NFP filters (Millipore) was nanofiltration the leaching solution 120 containing the ApoA-I ℓ (nano-filtration step).

<1-3> Rebuilding HDL

1,240 kg of soy phosphatidyl choline (SoyPC; PHOSPHOLIPID) and 715 g of sodium cholate (sodium cholate) were added to the nano-filtered ApoA-I solution, and stirred at 6 ° C. for 12 hours, using an ultrafiltration membrane of 10,000 Da. Dialysis with 500 L dialysis buffer (10 mM Tris-HCl, 10 mM NaCl, 1 mM EDTA, pH 7.5) and then again with 300 L of the dialysis buffer containing 1% (W / V) sucrose in succession. Dialysis concentrated to reconstitute HDL (reconstruction step).

1.35 kg sucrose was added to the 15 l concentrated rHDL solution, and stirred and sterilized and stored at 4 ° C.

The concentration of the rHDL produced above was 20 mg / ml, the turbidity was 12 NTU (Nephelometric Turbidity Units), and the produced rHDL was observed with an electron microscope (Carl Zeiss, LIBRA120). Have a size (FIG. 3A).

Example 2

The nanofiltration process was performed in the same manner as in <1-2> of Example 1 using 300 g of the pH precipitate obtained in <1-1> of Example 1.

780 g of POPC (1-palmitoyl 2-oleoyl phosphatidylcholine; Avanti Polar-lipid) and 700 g of sodium cholate were added to the nanoporous ApoA-I solution and stirred at 6 ° C. for 12 hours, followed by a 10,000 Da ultrafiltration membrane. Was dialyzed with 400 L of dialysis buffer, followed by dialysis to 250 L of the dialysis buffer containing 1% (W / V) sucrose in succession to reconstitute HDL.

1.17 kg of sucrose was added to the 13 l concentrated rHDL solution, followed by stirring and sterile filtration and storage at 4 ° C.

The concentration of rHDL produced above was 20.5 mg / ml, the turbidity was 5 NTU, and the produced rHDL was observed with an electron microscope and had a size of about 10 nm (FIG. 3B).

Using the ApoA-I purification method of the present invention can economically mass-purify high purity ApoA-I, and using the rHDL production method of the present invention using the purified ApoA-I, high purity and excellent viral safety rHDL Since it can be effectively mass-produced, it can be usefully used in the composition or formulation for the prevention or treatment of cardiovascular diseases, sepsis or anti-inflammatory, including hypertension, angina pectoris, myocardial infarction, stroke and cerebrovascular disease.

Claims (25)

ApoA-I-containing protein is extracted from the material containing ApoA-I (extraction step), the precipitant is treated to remove high molecular weight protein impurities (first precipitation step), and then the precipitant is treated secondly in the obtained solution. To precipitate the protein containing ApoA-I (second precipitation step), the precipitate was dissolved in a buffer solution and then treated with ethanol to remove high molecular weight protein impurities (ethanol precipitation step), the ethanol treated supernatant PH was adjusted to recover the precipitate (pH precipitation step), the precipitate was dissolved in a buffer or chaotropic solution and heat treated to inactivate the virus (virus inactivation step), and chromatographically separated ApoA-I. And (chromatography step) nanofiltration to remove the virus (nanofiltration step). The method of claim 1, The extraction step is characterized in that the ApoA-I-containing material is dissolved in urea, guanidine-HCl, alcohol or a mixture thereof, and then centrifuged or filtered to obtain a solution containing ApoA-I. Purification method. The method of claim 1, In the first precipitation step, a high molecular weight protein impurity is precipitated by adding polyethylene glycol (PEG) or ammonium sulfate as a precipitant to the solution obtained in the extraction step, followed by stirring, followed by centrifugation or filtration to obtain a solution containing ApoA-I. Purification method of ApoA-I, characterized in that to recover. The method of claim 1, The second precipitation step is to precipitate the protein containing ApoA-I by adding polyethylene glycol (PEG) or ammonium sulfate as a precipitant to the solution obtained in the first precipitation step, and then recovering the precipitate by centrifugation or filtration. The purification method of ApoA-I made into. The method of claim 1, The ethanol precipitation step is characterized in that the precipitate obtained in the secondary precipitation step is dissolved in a buffer of pH 7 or higher, and then ethanol is added to precipitate impurities, followed by centrifugation or filtration to recover a solution containing ApoA-I. Purification method of ApoA-I. The method of claim 1, The pH precipitation step is to adjust the pH of the solution obtained in the ethanol precipitation step to 5 to 7 to precipitate the protein containing ApoA-I, and then centrifuged or filtered to recover the precipitate, ApoA-I Purification method. The method of claim 1, The virus inactivation step is characterized in that the precipitate obtained in the pH precipitation step is dissolved in a buffer or chaotropic solution having a pH of 7 or more, and then heat-treated at 60 ° C. for 10 hours or more. The method of claim 1, Chromatography, characterized in that to purify the solution heat-treated in the virus inactivation step using an anion exchange resin, ApoA-I purification method. The method of claim 1, A method for purifying ApoA-I, wherein the substance containing ApoA-I is derived from plasma or is a recombinant ApoA-I culture solution. The method of claim 9, A method of purifying ApoA-I, characterized in that the substance comprising ApoA-I derived from plasma is Cohn fraction IV. The method of claim 3, wherein A method for purifying ApoA-I, characterized in that PEG is used at a concentration of 5-20% (W / V). The method of claim 4, wherein A method for purifying ApoA-I, characterized in that PEG is used at a concentration of 5-40% (W / V). The method of claim 5, wherein A method for purifying ApoA-I, characterized in that the protein concentration of the solution in which the precipitate is dissolved is 5 to 25 mg / ml. The method of claim 5, wherein A process for purifying ApoA-I, characterized in that 50 to 75% of ethanol is used. The method of claim 5, wherein A method for purifying ApoA-I, further comprising adding a salt or a metal ion such that the ionic strength of the solution after the ethanol is 2 to 20 mS / cm. The method of claim 6, Purification method of ApoA-I, characterized in that carried out at pH 5-7. The method of claim 7, wherein A method for purifying ApoA-I, characterized in that the chaotropic solution is 4-8 M guanidine-HCl solution or 4-8 M urea solution. Method for producing a reconstituted HDL comprising the step of adding the lipid and the surfactant to the purified ApoA-I solution according to the method of claim 1 at the same time and sufficiently stirred at low temperature, followed by dialysis using an ultrafiltration membrane. The method of claim 18, A method for producing reconstituted HDL, characterized in that the concentration of ApoA-I is 1 to 10 mg / ml. The method of claim 18, A method for producing reconstituted HDL, characterized in that the molar ratio of ApoA-I, lipid and surfactant is 1: 100 to 200: 150 to 250. The method of claim 18, A process for producing reconstituted HDL, wherein the lipid is phospholipid or synthetic phospholipid purified from soybean or egg. The method of claim 18, A method for producing reconstituted HDL, characterized in that the surfactant is a bile acid or bile salt comprising cholate, deoxycholate or ursodeoxycholate. The method of claim 18, Ultrafiltration membrane size of 1,000 to 100,000 Da, characterized in that the production method of reconstituted HDL. The method of claim 18, Method for producing reconstituted HDL, characterized in that the pH of the buffer used for dialysis is 6 to 10. The method of claim 18, The method for producing a reconstituted HDL, characterized in that it further comprises 5 to 15% disaccharides or 1 to 10% monosaccharides as an additive to increase the stability of the rHDL produced.

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