WO2011085598A1

WO2011085598A1 - Manufacture process for preparing high purity apoa-i from precipitate of plasma fraction iv.

Info

Publication number: WO2011085598A1
Application number: PCT/CN2010/077463
Authority: WO
Inventors: 黄凯; 何秋; 许必雄; 李春洲; 李军辉; 沈积慧; 郭颀然
Original assignee: 上海莱士血液制品股份有限公司
Priority date: 2010-01-15
Filing date: 2010-09-29
Publication date: 2011-07-21
Also published as: CN102127165A; CN102127165B

Abstract

The present invention provides a method for preparing high purity ApoA-I from the precipitate of plasma fraction IV, which including the following steps: dissolving the precipitate of plasma fraction IV, removing the diatomite and impurities by centrifugation, and collecting the supernatant; adding sodium chloride to said supernatant to precipitate ApoA-I protein, and obtaining ApoA-I precipitate by centrifugation; redissolving ApoA-I precipitate and filtrating it; separating the filtrate from the above procedure first by anion column chromatography then by hydrophobic column chromatography to obtain high purity ApoA-I solution.

Description

Production process for preparing high purity ApoA-I from plasma component four precipitation

The invention belongs to the technical field of biopharmaceuticals, and particularly relates to a production process for preparing high-purity apolipoprotein ApoA-I from four precipitation of plasma components. Background technique

Apolipoprotein AI (ApoA-I) is a major apolipoprotein of High Density Lip ₀ protein (HDL). It is a single polypeptide chain composed of 243 amino acid residues with a molecular weight of 28.3 kD. The main function of HDL is to participate in the reverse Cholesterol Transport (RCT), which removes and transports cholesterol from peripheral tissue cells to the liver for transformation and clearance, thus playing an important role in the prevention and development of atherosclerosis (AS). ApoA-I is the main bearer of HDL anti-AS function. At the same time, ApoA-I also has anti-inflammatory and anti-endotoxin functions, and is therefore one of the research priorities of lipid metabolism. In addition, because ApoA-I has the characteristics of liver targeting, it also has a good application prospect in targeted drug research.

At present, the commonly used preparation methods of ApoA-I include ultracentrifugation, organic solvent precipitation and high performance liquid chromatography. Although high purity Ap ₀ AI can be obtained, it has some obvious disadvantages: 1 The preparation amount is small, and it is not suitable for industrial use. Production; 2 low protein yield, ultra-centrifugation, organic solvent precipitation, column chromatography and other multi-step treatment, lost most of the ApoA-I in the preparation process; 3 high cost, requires expensive instruments such as ultracentrifuge; 4 security Poor, organic solvents such as ethanol, acetone, trichloroacetic acid, urea, etc., not only have physiological toxicity, but also flammable and explosive, unfavorable and safe production.

On the other hand, plasma component IV is the remainder of human plasma treated with Cohn low-temperature ethanol, and is discarded because it cannot be used. Its main protein components are albumin and beta lipoprotein. There are no reports on the preparation of ApoA-I using plasma fraction IV as raw material. Summary of the invention

The object of the present invention is to provide a high-purity apolipoprotein ApoA- which is suitable for industrial large-scale production, which is a raw material which is not fully utilized and is precipitated as a raw material, thereby providing a simple process, convenient operation, short production cycle and high yield. I purification process to make fuller use of plasma resources. The product prepared by the invention can be used for treating cardiovascular diseases such as atherosclerosis.

According to the physical and chemical properties of ApoA-I, the present invention obtains a suitable separation strip by adjusting the acidity and ionic strength of the suspension. The ApoA-I-rich protein precipitate is obtained by centrifugation, and then the precipitate is reconstituted. The anion exchange and hydrophobic chromatography column are used to select the appropriate loading and elution conditions to achieve high-efficiency separation of ApoA-I and heteroprotein. For purification purposes.

The present invention prepares high purity ApoA-I from a plasma fraction of four precipitates by two-step centrifugation plus two-step column chromatography. The key point of the technical scheme of the present invention is that the ApoA-I protein-rich precipitate is separated and centrifuged under suitable conditions and ApoA-I is purified by two-step column chromatography. In the centrifugation step, a protein precipitate rich in ApoA-I is obtained. In the ion exchange chromatography step, select the appropriate column conditions, ApoA-I and the heteroprotein are bound together to the column, and then elute with a suitable buffer to remove most of the impurities, and then concentrate with chlorination. The sodium solution elutes the column to obtain the preliminary purified Ap ₀ AI, then the hydrophobic column, some of the heteroprotein is removed with the flow through, and then further eluted with a suitable buffer to remove residual heteroprotein, and finally with WFI or alkaline solution. The hydrophobic column is eluted, and finally a high purity ApoA-I protein solution is obtained.

The production process of ApoA-I proposed by the present invention is shown in Figure 1

The method of the present invention for purifying high purity ApoA-I from component four comprises the following steps:

(1) The plasma component is dissolved in a buffer solution of pH 8.00-10.00, the diatomaceous earth and impurities are removed by centrifugation, and the supernatant is collected;

(2) ApoA-I protein was coagulated by adding sodium chloride to the supernatant, and centrifuged to obtain ApoA-I precipitate;

(3) reconstituting ApoA-I precipitate and filtering;

(4) The filtrate of the step (3) is separated by anion column chromatography and hydrophobic column chromatography to obtain a purified ApoA-I solution. In the purified ApoA-I solution, the purity of ApoA-I was over 95%.

After obtaining a high-purity ApoA-I protein solution, it can be prepared into a liquid preparation or lyophilized into a powder by a conventional method such as dialysis, concentration, virus inactivation, and filling or lyophilization.

In the step (1), the component four can be dissolved in a sodium acetate buffer solution having a pH of 8.00 to 10.00 at a low temperature (0 to 8 ° C), and sufficiently stirred to be sufficiently dissolved. Centrifugation removes diatomaceous earth and insoluble impurities.

In the step (2), the concentration of sodium chloride in the solution is 1-3 wt%, and then the pH is adjusted to 6.0-6.5, and the temperature is lowered to -1-rC to cause agglomeration of the ApoA-I protein.

The step (3) may reconstitute the ApoA-I precipitate by using a low temperature (0-10 ° C) WFI (water for injection) or a l-3 wt% sodium chloride solution having a pH of 8.0-9.0. It was then filtered through a 0.45 μηη filter.

In the step (4), the anion column chromatography uses a DEAE anion chromatography column, and the hydrophobic column chromatography uses a butyl hydrophobic column.

Further, the steps of the anion column chromatography are: adjusting the pH of the ApoA-I filtrate to 5.3-5.7, the ionic strength is 15-25 mM, then the DEAE anion chromatography column, and then eluting the DEAE column with the low ionic strength buffer. Thereafter, the column was eluted with a high-salt eluent, and the collected high-salt eluate was used to obtain a preliminary purified ApoA-I eluate. The low ionic strength buffer is preferably a Tris buffer having a pH of 5.00 - 5.10 and an ionic strength of 15 - 25 mM. The high salt eluent is preferably 0.9-1.1 M aqueous sodium chloride solution.

The steps of hydrophobic column chromatography are as follows: Adjust the pH of the initially purified ApoA-I eluate to 7.8-8.2 and then onto the butyl hydrophobic column, and then further elute with the low-salt eluent to remove residual heteroprotein, and finally use WFI or alkali. The hydrophobic solution was eluted with a solution to obtain an ApoA-I protein solution. The low salt eluate is preferably 0.01-0.05 M Tris buffer. The alkaline solution is preferably an aqueous NaOH solution having a pH of 10.30 to 11.30.

Features of the invention:

(1) Preparation of the process ApoA-I has high purity, purity of over 95%, and ApoA-I yield of 70%.

(2) The preparation process does not involve organic solvents, and the operation is safe and convenient.

(3) The results of the pilot test show that the combination of centrifugation and column chromatography to purify the Ap ₀ AI is easy to achieve process amplification, which is very suitable for industrial production. DRAWINGS

Figure 1: Block diagram of the production process of human plasma apolipoprotein ApoA-I

Figure 2: Results of SDS-PAGE electrophoresis of DEAE anion column chromatography

In the figure: 7 is the sample protein strip, 3 is the flow-through protein band, 2 low molecular weight standard protein, 1 is the heteroprotein eluate strip; 4, 5, 6 is the target protein eluate strip , the protein band indicated by the arrow is ApoA-I

Figure 3: Results of SDS-PAGE electrophoresis of Butyl column chromatography

In the figure: 1 is DEAE column chromatography ApoA-I protein eluent, 2 low molecular weight standard protein; 3, 4, 5, 6, 7, 8 is the target protein band purified by Butyl column chromatography; Protein band is ApoA-I

Figure 4: Western blot results

In the figure: 0 is albumin, 1-6 corresponds to l.Oug (Example 1), 0.25 ug (Example 1), 0.0625 ug (Example 2), lug (Example 2), 0.25 ug (Example 3), 0.0625ug (Example 3) purified ApoA-I protein, 7 and 8 correspond to l.Oul and 0.25ul of plasma component four solution

Primary antibody: anti-ApoA-I goat anti-, calbiochem Cat#: 178463, 1:2000

Secondary antibody: 驴Anti-Sheep secondary antibody, 1:5000 Detailed implementation

The following examples are given to further illustrate the invention, and it should be understood that the examples are not intended to limit the scope of the invention. Example 1: Preparation of ApoA-I sample (1) plasma component four precipitation dissolution and pretreatment

Weigh 4 kg of component 4 (wet weight), dissolve in 45 kg of sodium acetate buffer (control temperature is 0~8 °C), adjust the pH to about 9.00, stir well to dissolve, then use Beckman The diatomaceous earth and insoluble matter were removed by a centrifuge (7000 rpm), and the supernatant was collected.

(2) Centrifugation to obtain ApoA-I precipitate

Sodium chloride was added to the supernatant to a concentration of about 2% by weight, and then the pH was adjusted to 6.0-6.5, and the temperature was lowered to -1-1 V to precipitate apoA-I. Centrifuge at high speed, collect about 500 g of ApoA-I precipitate, and discard the supernatant.

(3) Reconstituted ApoA-I precipitate

500 g of ApoA-I precipitate was sufficiently dissolved in 5 kg of a 2 wt% sodium chloride solution at about 5 ° C, and then filtered through a 0.45 μηη filter.

(4) Column chromatography

The pH of the ApoA-I filtrate was adjusted to 5.3-5.7 with acetic acid-sodium acetate solution, the ionic strength was 20 mM, and the DEAE anion chromatography column was applied at a flow rate of 120 cm/h (the DEAE anion chromatography column volume was 3 liters, and the chromatography packing was DEAE). Sepharose FF), ApoA-I binds to the chromatographic column and some of the heteroprotein flows through. Then, the DEAE column was eluted with a pH 5.10, ionic strength of 20 mM acetic acid-sodium acetate buffer solution, and most of the heteroprotein was eluted; and the DEAE chromatography was washed with a high concentration of sodium chloride solution (about 1.0 M). Column, collect the preliminary purified ApoA-I protein solution; adjust the collected high salt eluate with O. l NaOH solution to pH 8.0 and then onto the butyl hydrophobic column, some of the heteroprotein is removed with the flow through, followed by low The salt eluate (0.02 M Tris buffer) was further eluted from the column to remove residual heteroprotein, and finally the hydrophobic column was eluted with ρΗΙΟ.30 aqueous NaOH solution to obtain ApoA-I protein solution. ApoA-I with a purity of 95% or more and a molecular weight of 28 KD was obtained by SDS-PAGE electrophoresis. The obtained protein was assayed by immunoturbidimetric assay and Western blot (see Figure 4), and the cell binding activity of ApoA-I was confirmed by binding to hepatocyte assay, and the obtained protein was finally confirmed to be ApoA-I protein.

(5) ApoA-I eluent post-treatment

The eluted ApoA-I was concentrated by ultrafiltration using a millipere pilot system, dialyzed against buffer, and then concentrated to about 7% (7 g/100 ml), mannitol to 5% or sucrose to 10 wt%, and the concentration of the protein was adjusted to 5 Wt % and adjust the pH to 7.00.

(6) Inactivation of Pasteurella virus

The above solution was incubated at 60 ° C for 10 hours to complete virus inactivation treatment.

(7) sterilization filtration and filling

The solution after virus inactivation was subjected to sterilization filtration, and the yield of ApoA-I was calculated to be 70% (ApoA-I and raw materials obtained) The weight ratio of ApoA-I in plasma fraction IV). Filling according to a certain specification, the ApoA-I liquid injection is prepared. Example 2

ApoA-I sample preparation

(1) plasma component four precipitation dissolution and pretreatment

Weigh the component 4 and precipitate 3 kg (wet weight), dissolve in sodium acetate buffer (control temperature is 0~8 °C), adjust the pH to 8.00, stir well to dissolve, then remove diatomaceous earth by centrifugation at 7000 rpm. And insoluble matter, collect the centrifugation supernatant.

(2) Centrifugation to obtain ApoA-I precipitate

Sodium chloride was added to the supernatant to a concentration of 1 wt%, and then the pH was adjusted to 6.0-6.5, and the temperature was lowered to -l-rc to cause agglomeration of ApoA-I. Centrifuge at high speed, collect about 300 g of ApoA-I precipitate, and discard the supernatant.

(3) Reconstituted ApoA-I precipitate

300 g of ApoA-I precipitate was sufficiently dissolved in a lwt% sodium chloride solution at 0 ° C, and then filtered through a 0.45 μηη filter.

(4) Column chromatography

The pH of the ApoA-I filtrate was adjusted to 5.3-5.7 with an acetic acid-sodium acetate solution, and the ionic strength was 15 mM. The DEAE anion chromatography column was applied at a flow rate of 120 cm/h, and ApoA-I and the hybrid protein were bound to the column. The impurity protein flows through. Then, the DEAE column was eluted with an acetic acid-sodium acetate buffer solution having an ionic strength of about 15 mM at a pH of about 5.10, and most of the heteroprotein was eluted; and the DEAE column was washed with a 0.9 M sodium chloride solution. The initially purified ApoA-I protein solution was collected; the collected high-salt eluate was adjusted to pH 7.8 with 0.1% NaOH solution, and then the butyl hydrophobic column was removed, and some of the heteroprotein was removed by flow through, followed by elution with low salt. The solution (0.01 M Tris buffer) was further eluted from the column to remove residual heteroprotein, and finally the hydrophobic column was eluted with a pH NaOH solution of NaOH to obtain an ApoA-I protein solution. ApoA-I with a purity of 95% or more and a molecular weight of 28 KD was obtained by SDS-PAGE electrophoresis. The obtained protein was determined by immunoturbidimetric assay and western blot (see Figure 4). The cell binding activity of ApoA-I was confirmed by binding to hepatocyte assay, and the obtained protein was finally confirmed to be ApoA-I protein.

(5) The post-treatment was the same as in Example 1, and the final yield of ApoA-I was 73%. Example 3

ApoA-I sample preparation

(1) plasma component four precipitation dissolution and pretreatment

Weigh 6 kg of component 4 (wet weight), dissolve in sodium acetate buffer (control temperature is 0~8 °C), adjust pH The value was 10.00, and the mixture was thoroughly stirred to dissolve, and then diatomaceous earth and insoluble matter were removed by centrifugation at 7000 rpm, and the centrifugation supernatant was collected.

(2) Centrifugation to obtain ApoA-I precipitate

Sodium chloride was added to the supernatant to a concentration of 3 wt%, and then the pH was adjusted to 6.0-6.5, and the temperature was lowered to -l-rc to cause agglomeration of ApoA-I. Centrifuge at high speed, collect about 600 g of ApoA-I precipitate, and discard the supernatant.

(3) Reconstituted ApoA-I precipitate

600 g of ApoA-I precipitate was sufficiently dissolved in a 3 wt% sodium chloride solution at about 10 ° C, and then filtered through a 0.45 μm filter.

(4) Column chromatography

The pH of the ApoA-I filtrate was adjusted to 5.3-5.7 with an acetic acid-sodium acetate solution, and the ionic strength was 25 mM. The DEAE anion chromatography column was applied at a flow rate of 120 cm/h (the DEAE anion chromatography column volume was 3 liters, and the chromatography packing was DEAE). Sepharose FF), ApoA-I binds to the chromatographic column and some of the heteroprotein flows through. Then, the DEAE column was eluted with a pH 5.00, 25 mM acetic acid-sodium acetate buffer solution, and most of the heteroprotein was eluted. The DEAE chromatography was washed with a high concentration sodium chloride solution (about 1.0 M). Column, collect the preliminary purified ApoA-I protein solution; adjust the collected high salt eluate with O. l NaOH solution to pH 8.0 and then onto the butyl hydrophobic column, some of the heteroprotein is removed with the flow through, followed by low The salt eluate (0.05 M Tris buffer) was further eluted from the column to remove residual heteroprotein, and finally the hydrophobic column was eluted with WFI to obtain an ApoA-I protein solution. ApoA-I with a purity of 95% or more and a molecular weight of 28 KD was obtained by SDS-PAGE electrophoresis. The obtained protein was determined by immunoturbidimetry and Western blot (see Fig. 4), and the hepatocyte test was carried out to verify the cell binding activity of ApoA-I, and finally the confirmed protein was ApoA-I protein.

(5) The post-treatment was the same as in Example 1, and the final yield of ApoA-I was 71%.

Claims

Claim

A method for purifying ApoA-I from plasma component four, comprising the steps of:

1) The plasma component is dissolved in a buffer of pH 8.00-10.00, the diatomaceous earth and impurities are removed by centrifugation, and the supernatant is collected;

2) The supernatant was added with sodium chloride to agglomerate the ApoA-I protein, and the ApoA-I precipitate was obtained by centrifugation;

3) reconstituting ApoA-I precipitate and filtering;

4) The filtrate of step 3) is separated by anion column chromatography and hydrophobic column chromatography to obtain a purified ApoA-I solution.

2. The method for purifying ApoA-I from plasma component IV according to claim 1, wherein in the step 1), the plasma component is dissolved in four steps: the plasma component is at 0-8. Dissolved in a sodium acetate buffer at pH 8. 00-10. 00.

3. The method for purifying ApoA-I from plasma component IV according to claim 1, wherein in the step 2), the method of agglomerating the ApoA-I protein by adding sodium chloride is: adding chlorine The concentration of sodium to sodium chloride in the solution is 1-3 wt%, and the pH is adjusted to 6. 0-6. 5, and the temperature is lowered to -1-1 ° C to precipitate the ApoA-I protein.

4. The method for purifying ApoA-I from plasma component IV according to claim 3, wherein the complex solution for reconstituting ApoA-I in step 3) is water for injection or 1-3 wt% of sodium chloride. The pH of the aqueous solution is 8. 0-9. 0, and the temperature is 0-10 ° C.

5. The method of purifying ApoA-I from the plasma fraction of the fourth component according to claim 4, wherein the step 3) reconstituted solution is filtered using a membrane of 0.45 Mffl.

The method for purifying ApoA-I from plasma component IV according to any one of claims 1 to 5, wherein in step 4), the anion column chromatography uses a DEAE anion chromatography column. The hydrophobic column chromatography uses a butyl hydrophobic column.

7. A method of purifying ApoA-I from plasma fraction IV as claimed in claim 6, wherein

The step of the anion column chromatography is as follows: adjusting the 11 (to-1) filtrate of 11 to 5. 3-5. 7, the ionic strength is 15-25 mM, then the upper DEAE anion chromatography column, and then washing with a low ionic strength buffer After de-deioning the column, the column is eluted with a high-salt eluent to obtain a preliminary purified ApoA-I eluate;

The step of the hydrophobic column chromatography is: adjusting the pH of the initially purified ApoA-I eluate to 7.8-8. 2, then butyl hydrophobic column, and then further eluting with the low salt eluate to remove residual impurity protein. Finally, the hydrophobic column is eluted with water for injection or alkaline solution to obtain a solution of ApoA-I protein.

The ionic strength buffer is 15-25 mM Tris buffer. The low ionic strength buffer is PH5. 00-5. 10 ionic strength is 15-25 mM Tris buffer. Liquid; the high salt eluent is

The aqueous solution of sodium chloride is 0. 9-1. The aqueous solution of sodium hydroxide is 0. 01-0. 05M Tris buffer; the alkaline solution is NaOH solution of ρΗΙΟ. 3-11.

The method for purifying ApoA-I from plasma component IV according to any one of claims 1 to 8, wherein a high purity ApoA-I protein solution is obtained, followed by dialysis and concentration by a conventional method. After the virus is inactivated and filled or lyophilized, the liquid preparation is prepared or lyophilized into a powder.