KR20070042276A - Method for manufacturing purified apolipoprotein a-i product from human plasma - Google Patents

Abstract

The present invention relates to a method for separating and purifying apolipoprotein AI from human plasma, wherein cohn's fraction IV paste fractionated from human plasma is urea, alcohol, guanidine hydrochloride. dissolving in guanidine hydrochloride or a mixture extract thereof, and then removing the filter aid to obtain a supernatant by centrifugation or filtration. A primary impurity removal step of adding PEG (polyethylene glycol) to the supernatant and stirring to precipitate plasma protein impurities of high molecular weight, followed by centrifugation or filtration to obtain a supernatant; A secondary impurity removal step of precipitating plasma protein impurities of high molecular weight by mixing alcohols having an alcohol concentration of 50 to 70% in the supernatant and stirring them at -10 to 10 ° C; A third impurity removal step of heating and cooling the cold alcohol-treated mixture and centrifuging or filtration to obtain a supernatant; Gel-filtration or dialysis the supernatant to remove alcohol; And a lipid removal step of removing lipid components through an ion exchange column to provide high purity apolipoprotein A-I.

Apolipoprotein A-I, plasma, cohn's fraction method, corn fraction IV, cold ethanol method, heat treatment

Description

Method for manufacturing purified apolipoprotein A-I product from human plasma}

1 is SDS-PAGE gel electrophoresis results of Examples 1, 6 and 7 of the present invention. Lane 1: loading solution, Lane 2: 50% ethanol + heat treatment (Example 6), Lane 2: 60% ethanol + heat treatment (Example 1), Lane 3: 70% ethanol + heat treatment (Example 7)

Figure 2 shows the results of SDS-PAGE gel electrophoresis of apolipoprotein A-I purified by treatment with cold ethanol without heat treatment. Lane 1: loading solution, Lane 2: 50% ethanol precipitation (Experimental Example 1-1), Lane 2: 60% ethanol precipitation (Experimental Example 1-2), Lane 3: 70% ethanol precipitation (Experimental Example 1-3)

3 shows the results of SDS-PAGE gel electrophoresis of apolipoprotein A-I purified without PEG precipitation. Lane 1: loading solution, Lane 2: 50% ethanol + heat treatment (Experimental Example 2-3), Lane 2: 60% ethanol + heat treatment (Experimental Example 2-3), Lane 3: 70% ethanol + heat treatment (Experiment 2 -3)

4 is a result of dialysis and concentration of purified apolipoprotein A-I of Example 7, Experimental Example 1-3 and Experimental Example 2-3 at 70% ethanol concentration, followed by SDS-PAGE gel electrophoresis.

Figure 5 is the result of removing the lipid (lipid) with DEAE-Sepharose ion exchange column. A: laoding solution B: non-adsorbent C: eluent D: final concentrate

Figure 6 is a result of SDS-PAGE gel electrophoresis by reducing or non-reducing the non-adsorbed liquid, eluent, concentrated liquid and the like filtered by DEAE-Sepharose ion exchange column. Lane 1: loading solution, Lane 2: non-adsorbent, Lane 3: washed, Lane 4: eluent-1, Lane 5: eluent-2, Lane 6: control (Apolipoprotein A-I) Sigma

The present invention relates to a method for separating useful bioproteins from human plasma from human plasma, and more particularly, from atherosclerosis and cardiovascular diseases from cohn's fraction IV derived from plasma. The present invention relates to a method for isolating and purifying apolipoprotein AI used as a therapeutic agent for cardiovascular disease, sepsis and the like.

The main function of lipoproteins in plasma is to carry lipids such as cholesterol and triglycerides (TG).

Since fats such as cholesterol are not soluble in water, the problem of how to dissolve it in transport plasma is solved by the combination of nonpolar fats such as TG and bipolar fats (phospholipids and cholesterol), and lipoproteins are mixed with water. To be.

Lipoproteins are divided into four main groups. : Chylomicron (CM), very low density lipoprotein (VLDL), low density lipoprotein (LDL), high density lipoprotein (HDL)

Among them, high density lipoprotein (HDL) plays a direct role in removing cholesterol from peripheral tissues and transporting it to the liver or other lipoproteins, where cholesterol is metabolized in the liver.

The major proteins of high-density lipoproteins are composed of Apolipoprotein A-I (70%) and Apolipoprotein A-II (20%).

The protective role of high density lipoproteins has been confirmed in many studies. Recent studies have focused on apolipoprotein A-I, a major protein of high-density lipoproteins. In particular, it was found that the level of Apolipoprotein A-I is associated with the risk of coronary artery disease.

Conventionally, ultracentrifugation has classically been used as a method of separating high-density lipoproteins from plasma. However, this method has been limited to separating small amounts at the laboratory level. Cohn and Oncley Fraction III (or Kistler and Nitschmann Precipitate B) was used as the main initial material for commercial mass production. Cohn fraction IV is also known to be rich in high density lipoproteins.

Several methods have been proposed for purifying apolipoprotein A-I from plasma.

The most common method is to use an ultracentrifuge to separate high density lipoproteins and then to separate the apolipoprotein A-I from the high density lipoproteins. As a method for separating apo lipoprotein A-I from the high density lipoprotein, various separation methods such as an organic solvent extraction method have been used. However, ultracentrifugation is a very time consuming method and is not suitable for mass production.

In addition, the method for producing apolipoprotein AI from plasma (Ross SE et al, Rapid chromatographic purification of apolipoprotein AI and A-II from human plasma, Analytical Biochemistry 149, p. 166-168 (1985)) and Gel-filtration HPLC (Tricerri A. et. al. A rapid and efficient procedure for the purification of human apolipoprotein using gel-filtration HPLC, IJBC, 1, p. 159-166 (1994)) was studied.

As another separation and purification method, the method of using the fraction separated by the ethanol fractionation method as an initial material was examined. (Peisch et.al. A purification method for apolipoprotein AI and A-II, Analytical Biochemistry, 178, p. 301-305 ( 1989).

In EP-A0329605, Rotkreuzstiftung Zentrallaboratorium Blustspendedienst SRK and Lerch et.al. purify apolipoproteins from fractions of human plasma containing lipoproteins, and precipitation B and fraction IV can produce apolipoprotein A during cold ethanol fractionation. It has been shown to be an initial material. High concentrations of alcohol and organic solvent were used to precipitate impure materials, and guanidine hydrochloride was removed by gel filtration. Anion exchange column process was used to pass apolipoprotein A and bind impurities.

In JP-A-08003198, the Chemo-Sero-Therapeutics Research Institute purified apolipoproteins by reacting phosphorus with low concentrations of alcohol through a hydrophobic column.

In WO-A-93 / 12143, Pharmacia & Upjohn (formerly Kabi Pharmacia AB) isolated and purified apolipoprotein A-IM produced from plasma using apolipoprotein A-IM and recombinant DNA technology. High density lipoproteins were separated from the plasma and purified dimers by gel filtration several times, and the monomers were separated using Thiopropyl-SEPHAROSE to make dimers.

Various fractionation methods have been used to separate and purify albumin and globulin from human plasma. The most commonly used method is cold ethanol fractionation method such as Cohn or Cohn-Oncley process.

Cold ethanol fractionation (cold ethanol precipitation) is a method used in the production of plasma fractions by collecting the plasma from a number of donor blood. This method collects a large amount of plasma and adjusts the protein concentration, alcohol concentration, pH, salt concentration (ion intensity), and temperature of the liquid at a low temperature of about -5 ° C to take advantage of the difference in solubility of albumin and gamma globulin. It is a process to produce blood products such as anti-hemophilic globulin and fibrinogen.

Cold ethanol fractionation process is a method using the principle that the specific protein is not dissolved in each fractionation process precipitated proteins are separated by centrifugation or filtration. In cold ethanol fractionation, alcohol is used to precipitate proteins at low temperatures. Usually the alcohol concentration is gradually increased from 0% to 40% and the temperature is varied from 0 ° C to -10 ° C. The precipitate of the process gives five fractions, represented by fractions I to V.

For example, fresh human plasma or frozen human plasma obtained by hemocytosis collection method can be separated by cold sedimentation method and DEAE-Sephadex adsorption method. (Blood coagulation factor) and the supernatant I, and then only the supernatant I was taken and fractionated into fractions II + III (immunoglobulin) and supernatant II + III by cold ethanol precipitation. The supernatant II + III is further fractionated into fraction IV and supernatant IV by cold ethanol precipitation. Fraction IV is discarded and supernatant IV is separated into fraction V by cold ethanol precipitation. Fraction V is used as a source of albumin.

In Cohn fractionation, the fraction containing lipoprotein is separated into fraction III and fraction IV. Currently the above fractions are being discharged as waste.

The present invention provides a method for separating high apolipoprotein AI, which is used as a therapeutic agent for atherosclerosis, cardiovascular disease, sepsis, etc. by using a cohn fraction IV discharged as waste in the albumin manufacturing process as an initial material. The purpose is.

Hereinafter, the apolipoprotein A-I separation and purification method of the present invention will be described in detail.

In the apolipoprotein AI separation and purification method of the present invention, the human plasma corn fraction IV is washed to remove impurity proteins such as albumin, and the filter aid is removed with urea (alcohol), guanidine hydrochloride, and the like. In a conventional method of purifying apolipoprotein AI by precipitating and removing high molecular weight other protein impurities with a precipitating agent, and then removing lipids by chromatography,

Cold alcohol is mixed with the filtrate or the supernatant after the PEG precipitation treatment and stirred at -10 ~ 10 ℃ to precipitate other high molecular weight impurities again, and the cold alcohol treated mixture is heat-treated at 60 ~ 70 ℃ It can be achieved by including more.

More specifically, the apolipoprotein A-I separation and purification method of the present invention,

Cohn's fraction IV paste fractionated from human plasma was dissolved in urea, alcohol, guanidine hydrochloride, or mixtures thereof, followed by centrifugation or filter press. Filter aid removal step to obtain a supernatant;

Primary impurity removal step of adding PEG (polyethylene glycol) to the supernatant and stirring to precipitate plasma protein impurities of high molecular weight, followed by centrifugation or filtration to obtain supernatant;

A secondary impurity removal step of precipitating plasma protein impurities of high molecular weight by mixing alcohols having an alcohol concentration of 50 to 70% in the supernatant and stirring them at -10 to 10 ° C;

A third impurity removal step of heating and cooling the cold alcohol-treated mixture and centrifuging or filtration to obtain a supernatant;

Gel-filtration or dialysis the supernatant to remove alcohol; And it comprises a lipid removal step of removing the lipid component through the ion exchange column.

When a precipitate containing lipoproteins is formed by cold ethanol fractionation by the cone fractionation method, it is mainly separated by a filter press. At this time, it is easier to filter by adding a filter aid such as supercel. Therefore, corn fraction IV generally contains not only lipoproteins but also filter aids, and other proteins such as albumin are precipitated together. The present invention is completed by removing these filtration aids and other protein impurities.

Prior to removing the filter aid, a washing process is first introduced to remove impurities protein such as albumin from corn fraction IV. As the washing buffer, sodium chloride, organic acids such as alcohol and citric acid, minerals such as phosphate may be used, and the washing temperature is preferably performed at 3 ° C. to 20 ° C. It is not limited. The washing buffer was added to the cone fraction IV to adjust the pH appropriately, and then suspended using a stirrer. The suspension was filtered using a filter press to obtain a precipitate, or centrifuged to give a supernatant. Discard the various plasma protein precipitates including apolipoprotein AI can be obtained. At this time, apolipoprotein A-I is hardly removed and is present in the precipitate more than 90%.

Corn fraction IV containing lipoprotein contains a filter aid and binds with strong binding force. Therefore, a process for completely extracting the filter aid is required. As the extract, urea, guanidine hydrochloride, alcohol, and the like may be used. More preferably, urea is used. Extraction temperature is preferably carried out at about 4 ℃ ~ 20 ℃, but is not limited thereto. The urea (Urea) concentration is preferably 4 ~ 8M, and after completely dissolving in the extraction solution, the stirring is performed for about 1 to 5 hours, separated by centrifugation or filtration to obtain a supernatant. It is confirmed that apolipoprotein A-I is extracted completely along with a myriad of plasma proteins.

In addition, the same result can be confirmed in an experiment in which the recycled urea extract was recycled for 1 to 5 hours without recovering the precipitate of the cone fraction IV washing process.

Apolipoprotein A-I is a monomer of about 28,000 Da and is a relatively low molecular weight protein in cone fraction IV. Therefore, separating and purifying apolipoprotein A-I in plasma may be a method of removing impurity proteins having high molecular weight.

As a method of separating and purifying proteins, precipitation methods using various precipitation enhancers have been developed. Precipitation methods using PEG and ammonium sulfate are mainly used. PEG is classified by PEG 4000, PEG 6000, PEG 8000, PEG 20000, etc., preferably PEG 4000 but is not limited thereto.

The concentration of PEG to remove high molecular weight plasma protein is preferably about 10 to 40%, added to the extract containing apolipoprotein AI, stirred for about 1 to 5 hours, and carried out at about 4 to 20 ° C. desirable. The supernatant separated from the mixture stirred with PEG by centrifugation or filtration is first removed a large amount of high molecular weight plasma protein.

Cold ethanol precipitation is performed in the same or similar manner as the cone fractionation method to remove the high molecular weight plasma protein remaining after the first impurity removal. The alcohol concentration is preferably about 50 to 70%, and the precipitation temperature is preferably carried out at a temperature change of -10 to 10 ° C. Alcohol is used as a precipitating agent at low temperature, and impurities of plasma protein impurities of high molecular weight other than apolipoproteins are generated and secondary impurities are separated. Experimental results showed that the purification effect is excellent at alcohol concentration of 65 ~ 70%.

In addition, an experiment is performed to separate and purify the plasma protein by heating the solution containing alcohol. The alcohol concentration is 60 ~ 70%, temperature is 60 ~ 70 ℃ 30 ~ 120 minutes, preferably 30 ~ 60 minutes heat treatment, then ice-cooled and separated by centrifugation or filtration method and the supernatant separated 3 Secondly, large amounts of high molecular weight plasma protein impurities are removed. Since the heat treatment is heat-treated at 60 ℃ or more has the additional effect of reducing the risk of viral infection by inactivating the plasma infected AIDS and hepatitis B virus.

Gel-filtration and ultrafiltration using membranes may be used to remove the alcohol components used for the impurity extraction. Membranes are produced by PALL, Millipore, Sartorius, and the like, preferably using 10KD pores. When using a dialysis method using a membrane, the apolipoprotein AI can be directly escaped by diluting 3-10 times in advance with a dialysis buffer, thereby recovering almost all proteins and efficiently removing alcohol components. Can be.

Since the apolipoprotein A-I solution separated and purified above has many lipid components generated in the extraction process, additional introduction of a lipid removal process is required. The method mainly used for removing lipids is to remove lipids by precipitating proteins using organic solvents such as ethanol, diethyl ether, hexane, isopropanol, etc. (organic solvent extraction), organic solvent components are filled with nitrogen and blown away, and dissolved with guanidine hydrochloride and then dialyzed. However, this method is possible at the laboratory level and is relatively unsuitable for production scale.

In the present invention, it is preferable to use an ion exchange column as a lipid removal step, and more preferably, Q-Sepharose and DEAE-Sepharose, which are anion exchange columns, are used. Lipids do not bind to the column and exit as a non-adsorbent, and all of the apolipoprotein A-I components bind to the column and elute. Examination of the eluate confirms that no lipids are present.

The apolipoprotein A-I solution separated and purified according to the present invention has a purity of 90% or more, and removes both lipid and alcohol components.

The present invention will be described in detail by way of examples. The following examples are merely examples for describing the present invention, and the scope of the present invention is not limited to the following examples.

[ Example  One]

1. Cone Fraction  wash( Washing )

6 g of Cohn's fraction IV paste was placed in a 100 ml test tube, and then 50 ml of a washing buffer consisting of 0.07 M Citric Acid and monohydrate 0.08 M Na ₂ HPO ₄ · H ₂ 0 was added to adjust the pH to 4.0 ± 0.05. It was then maintained for about 2 hours using a rocking machine while maintaining 3 ~ 5 ℃. The suspension was centrifuged at 3500 RPM for 10 minutes using a centrifuge. The supernatant was discarded to give a precipitate.

2. Urea  extraction( Extraction )

The washed precipitate was placed in a 100 ml test tube, and then 50 ml of 8M Urea was added and suspended for about 2 hours using a rocking machine while maintaining 3 to 5 ° C. The suspension was centrifuged at 3500 RPM for 30 minutes at 10 ° C. to obtain a supernant containing apolipoprotein.

3. PEG treatment

Since apolipoprotein A-I is a low molecular weight protein of about 28,000 Da, PEG (polyethyleneglycol) was treated to precipitate and remove high molecular weight plasma protein impurities contained in the supernatant.

30 ml of the obtained supernatant was added to a test tube, 6.0 g of PEG 4000 was added to a 20% PEG concentration, and then stirred at about 20 ° C. for about 2 hours. It was observed that turbidity increased as the stirring proceeded, and no spin bar was observed after 2 hours of stirring. The stirred mixture was left at room temperature and refrigerated to observe the change visually. No particular change was found in the first 30 minutes, but after 60 minutes, about 1 cm of precipitation occurred in the room temperature mixture and no precipitation was observed in the cold standing.

After 3 hours of standing, centrifugation was performed at 3500 rpm and 4 ° C for about 30 minutes.

32.5 ml of supernatant and 1.34 g of paste were fractionated at room temperature and 32.0 ml of supernatant and 3.61 g of paste.

The supernatant was refrigerated for one week and a slight white precipitate was observed.

4. Cold Ethanol  Sedimentation

A deol fraction was performed in cold to remove lipoprotein impurities from the PEG-treated supernatant. 60% ethanol was added to the PEG-treated supernatant, and then the temperature was lowered and gradually precipitated at -10 to 10 ° C. This is again carried out the cohn's fraction for plasma.

5. Heating treatment ( heat treatment )

The mixed solution fractionated with ethanol under low temperature was purified by denaturation of plasma protein through heat treatment. The concentration of ethanol was about 60%, and the temperature was heated at about 65 ° C. for at least 30 minutes, followed by ice cooling. Since the heat treatment is heat-treated at 60 ℃ or more has the additional effect of reducing the risk of viral infection by inactivating the plasma infected AIDS and hepatitis B virus.

6. Ethanol Removal

Ultrafiltration was performed using a membrane to remove ethanol used for the impurity extraction. The pore of the membrane was about 10 KD, and by diluting about 5 times with the dialysis buffer, almost all proteins were recovered and ethanol was efficiently removed.

[ Example  2 to 5]

In the same manner as in Example 1, in the PEG treatment step, the concentration of PEG was 10% (PEG 4000 3.0g), 15% (PEG 4000 4.5g), 25% (PEG 4000 7.5g), 30% (PEG 4000 9.0 g) and then stirred at about 20 ° C. for about 2 hours.

Precipitation experiment results according to the concentration change of PEG 4000 carried out in Examples 1 to 5 are shown in Table 1 below.

Item 10% 15% 20% 25% 30% Remarks object 8M Urea Extract Filtrate Liquid amount (ml) 30 ml PEG addition amount (g) 3.0 4.5 6.0 7.5 9.0 Liquid temperature at addition (℃) 20 ℃ Stirring time 2 hours Visual inspection after stirring Spin bar confirmed Spin bar not visible and becomes more turbid at 30% politics Room temperature cold storage Room temperature cold storage Room temperature cold storage Room temperature cold storage Room temperature cold storage Observed after 30 minutes Observed after 60 minutes - - - - 1 cm - 0.5 cm - 0.2 cm - Settling start 90 minutes   Room temperature-Sediment sinks down over time Refrigeration-Sediment forms but does not sink down 10% After centrifugation at room temperature, refrigeration, the yellow substance does not sink and floats in the supernatant. Same as 2 experiments 120 minutes 150 minutes 180 minutes Centrifugal conduct 3,500 RPM, 4 ° C, 30 minutes Supernatant (ml) - - 32.5 32.5 32.5 32.0 33.0 33.0 35.0 35.0 Increased by the amount of PEG added Paste (g) - - 0.72 0.69 1.34 3.61 2.02 1.91 1.78 2.17 15% high temperature paste

As shown in the above table, it can be seen that precipitation is efficiently performed at a PEG concentration of 15 to 30%, and in particular, the amount of paste is maximized when the PEG concentration is 15% refrigerated.

[ Example  6]

In order to confirm the impurity protein precipitation according to the change in the concentration of cold ethanol, the same method as in Example 1, except that 50% ethanol was added to the PEG-treated supernatant in the cold ethanol precipitation step, the temperature was lowered to -10 It gradually cooled to ˜10 ° C.

[ Example  7]

In the same manner as in Example 1, 70% ethanol was added to the PEG-treated supernatant in the cold ethanol precipitation step, and then the temperature was lowered to be gradually cold precipitated at -10 to 10 ° C.

SDS-PAGE to confirm the purity of the apolipoprotein AI purified according to Example 1 (cold ethanol concentration 60%), Example 6 (cold ethanol concentration 50%), Example 7 (cold ethanol concentration 70%) Gel electrophoresis was performed.

1 is SDS-PAGE gel electrophoresis results of Examples 1, 6 and 7.

Here, Lane 1: loading solution, Lane 2: 50% ethanol + heat treatment (Example 6), Lane 2: 60% ethanol + heat treatment (Example 1), Lane 3: 70% ethanol + heat treatment (Example 7)

As shown in FIG. 1, a thick band was formed at the molecular weight of the apolipoprotein A-I (about 28 kDa), and it was confirmed that most other proteins having a large amount of high molecular weight were removed. In particular, almost all other proteins were removed at an ethanol concentration of 70% (Example 7).

[ Experimental Example  One]

In order to compare the effects of the heat treatment of cold ethanol in Examples 1 to 7, this was carried out with the heat treatment steps omitted in Examples 1, 6 and 7.

Figure 2 shows the results of SDS-PAGE gel electrophoresis of apolipoprotein A-I purified by treatment with cold ethanol without heat treatment.

Lane 1: loading solution, Lane 2: 50% ethanol precipitation (Experimental Example 1-1), Lane 2: 60% ethanol precipitation (Experimental Example 1-2), Lane 3: 70% ethanol precipitation (Experimental Example 1-3)

As shown in Figure 2, it can be seen that other proteins of high molecular weight are removed to some extent, in particular, it can be seen that a significant amount of high molecular weight impurity protein is removed at an ethanol concentration of 70% (Experimental Example 1-3). However, as compared with Example 7 of FIG. 1, it can be seen that a large amount of impure protein still remains, thereby reducing the purification effect.

[ Experimental Example  2]

In order to compare the high molecular weight impurity protein removal effect by the PEG precipitation treatment in Examples 1 to 7, this was carried out without the PEG precipitation step in Examples 1, 6 and 7.

3 shows the results of SDS-PAGE gel electrophoresis of apolipoprotein A-I purified without PEG precipitation.

Lane 1: loading solution, Lane 2: 50% ethanol + heat treatment (Experimental Example 2-3), Lane 2: 60% ethanol + heat treatment (Experimental Example 2-3), Lane 3: 70% ethanol + heat treatment (Experiment 2 -3)

As shown in Figure 3, the ethanol concentration of 50%, 60% almost no other high molecular weight protein was removed, it can be seen that the ethanol concentration of 70% (Experimental Example 2-3) to some extent removed. However, as compared with Example 7 of FIG. 1, it can be seen that a large amount of impure protein still remains, thereby reducing the purification effect.

4 is a result of dialysis and concentration of purified apolipoprotein A-I of Example 7, Experimental Example 1-3 and Experimental Example 2-3 at 70% ethanol concentration, followed by SDS-PAGE gel electrophoresis.

As shown in Figure 4, the apolipoprotein purification method of the present invention has been shown to be able to remove significantly more impurity protein than without PEG treatment or heat treatment.

[ Example  8] Delipidation delipidation )

The apolipoprotein A-I solution separated and purified in the above example has many lipid components generated in the extraction process, and thus, an additional lipid removal process has been introduced.

In the present invention, it is preferable to use an ion exchange column as a lipid removal step, and more preferably, Q-Sepharose and DEAE-Sepharose, which are anion exchange columns, are used. Lipids do not bind to the column and exit as a non-adsorbent, and all of the apolipoprotein A-I components bind to the column and elute. Examination of the eluate confirmed that no lipid component was present.

Equilibrate DEAE-Sepharose (2.6 × 10.0 cm) with 30 mM Tris-HCl buffer (pH 7.4), then inject the sample into the column at 1 ml / min flow rate and in the same buffer containing 0.1 M NaCl (0.1 M NaCl). in 30 mM Tris buffer pH 7.4) until the absorbance of 280 nm was nearly zero. After washing it was eluted with the same buffer (1.0 M NaCl in 30 mM Tris buffer pH 7.4) containing 1 M NaCl.

Figure 5 is the result of removing the lipid (lipid) with DEAE-Sepharose ion exchange column.

A: laoding solution B: non-adsorbent C: eluent D: final concentrate

Figure 6 shows the results of SDS-PAGE gel electrophoresis by reducing or non-reducing the non-adsorbed liquid, washing liquid, eluent and the like filtered by DEAE-Sepharose ion exchange column. Lane 1: loading solution, Lane 2: non-adsorbent, Lane 3: wash, Lane 4: eluent-1, Lane 5: eluent-2, Lane 6: control (Apolipoprotein A-I)

As shown in Figure 6, it can be confirmed that most of the other high molecular weight proteins are removed.

In the present invention, the urea treatment and PEG treatment are performed using a cohn fraction IV discharged as waste in the albumin manufacturing process as an initial material, and the impure protein is removed to some extent, cold at a low temperature of -10 to 10 ° C. Apolipoprotein used as a therapeutic agent for atherosclerosis, cardiovascular disease and sepsis by removing alcohol once more by removing high molecular weight impurity protein and heating at about 60 ~ 70 ℃ It provides the effect of separating and purifying AI with high purity.

On the other hand, the heat treatment is heat treated at 60 ° C or more, thereby inactivating the plasma infected AIDS and hepatitis B virus provides an effect of reducing the risk of virus infection.

Claims (8)

Human plasma corn fraction IV was washed to remove impurities such as albumin, and filter aids were removed with urea (alcohol), guanidine hydrochloride, etc. In the method of purifying the apo lipoprotein AI by removing the lipid by precipitation, the lipid (lipid) by chromatography, Cold alcohol is mixed with the filtrate or the supernatant after the PEG precipitation treatment and stirred at -10 ~ 10 ℃ to precipitate other high molecular weight impurities again, and the cold alcohol treated mixture is heat-treated at 60 ~ 70 ℃ Apolipoprotein AI separation and purification method comprising a. The method of claim 1, wherein the cold alcohol concentration is 50 to 70% Apolipoprotein A-I separation and purification method characterized in that. Cohn's fraction IV paste fractionated from human plasma was dissolved in urea, alcohol, guanidine hydrochloride, or mixtures thereof, and then centrifuged or filtered press. Filter aid removal step to obtain a supernatant; A primary impurity removal step of adding PEG (polyethylene glycol) to the supernatant and stirring to precipitate plasma protein impurities of high molecular weight, followed by centrifugation or filtration to obtain a supernatant; A secondary impurity removal step of precipitating plasma protein impurities of high molecular weight by mixing alcohols having an alcohol concentration of 50 to 70% in the supernatant and stirring them at -10 to 10 ° C; A third impurity removal step of heating and cooling the cold alcohol-treated mixture and centrifuging or filtration to obtain a supernatant; Gel-filtration or dialysis the supernatant to remove alcohol; And Apolipoprotein A-I separation and purification method comprising the step of removing the lipid component through the ion exchange column. 4. The method for separating and purifying apolipoprotein A-I according to claim 3, wherein the urea concentration is 4 to 8 M and carried out at 4 to 20 ° C. The method for separating and purifying apolipoprotein A-I according to claim 3 or 4, wherein the PEG concentration is 10 to 40% (w / w) and is performed at 4 to 20 ° C. The method for separating and purifying apolipoprotein A-I according to claim 3 or 4, wherein the heat treatment is performed at 60 to 70 ° C. 5. The method of claim 3 or 4, wherein the pores of the dialysis membrane are about 10 KD. The method for separating and purifying apolipoprotein A-I according to claim 3 or 4, wherein the ion exchange column is DEAE-Sepharose or Q-Sepharose.

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