WO2011085598A1 - Procédé de fabrication pour préparer apoa-i de pureté élevée à partir d'un précipité de fraction iv de plasma - Google Patents

Procédé de fabrication pour préparer apoa-i de pureté élevée à partir d'un précipité de fraction iv de plasma Download PDF

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WO2011085598A1
WO2011085598A1 PCT/CN2010/077463 CN2010077463W WO2011085598A1 WO 2011085598 A1 WO2011085598 A1 WO 2011085598A1 CN 2010077463 W CN2010077463 W CN 2010077463W WO 2011085598 A1 WO2011085598 A1 WO 2011085598A1
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apoa
solution
protein
column
precipitate
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PCT/CN2010/077463
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English (en)
Chinese (zh)
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黄凯
何秋
许必雄
李春洲
李军辉
沈积慧
郭颀然
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上海莱士血液制品股份有限公司
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Publication of WO2011085598A1 publication Critical patent/WO2011085598A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/775—Apolipopeptides
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P9/00—Drugs for disorders of the cardiovascular system
    • A61P9/10—Drugs for disorders of the cardiovascular system for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis

Definitions

  • 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 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).
  • RCT reverse Cholesterol Transport
  • ApoA-I is the main bearer of HDL anti-AS function.
  • ApoA-I also has anti-inflammatory and anti-endotoxin functions, and is therefore one of the research priorities of lipid metabolism.
  • ApoA-I has the characteristics of liver targeting, it also has a good application prospect in targeted drug research.
  • the commonly used preparation methods of ApoA-I include ultracentrifugation, organic solvent precipitation and high performance liquid chromatography.
  • 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.
  • 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.
  • 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.
  • the centrifugation step a protein precipitate rich in ApoA-I is obtained.
  • 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 method of the present invention for purifying high purity ApoA-I from component four comprises the following steps:
  • 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;
  • ApoA-I protein was coagulated by adding sodium chloride to the supernatant, and centrifuged to obtain ApoA-I precipitate;
  • the filtrate of the step (3) is separated by anion column chromatography and hydrophobic column chromatography to obtain a purified ApoA-I solution.
  • the purity of ApoA-I was over 95%.
  • a high-purity ApoA-I protein solution 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.
  • 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.
  • 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 ⁇ filter.
  • WFI water for injection
  • l-3 wt% sodium chloride solution having a pH of 8.0-9.0. It was then filtered through a 0.45 ⁇ filter.
  • the anion column chromatography uses a DEAE anion chromatography column
  • the hydrophobic column chromatography uses a butyl hydrophobic column.
  • 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.
  • Figure 1 Block diagram of the production process of human plasma apolipoprotein ApoA-I
  • 7 is the sample protein strip
  • 3 is the flow-through protein band
  • 1 is the heteroprotein eluate strip
  • 4 is the target protein eluate strip
  • the protein band indicated by the arrow is ApoA-I
  • 1 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
  • 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
  • Example 1 Preparation of ApoA-I sample (1) plasma component four precipitation dissolution and pretreatment
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the above solution was incubated at 60 ° C for 10 hours to complete virus inactivation treatment.
  • 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.
  • 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.
  • 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.
  • 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.

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Abstract

La présente invention concerne un procédé de préparation de ApoA-I de pureté élevée à partir du précipité d'une fraction IV de plasma, incluant les étapes suivantes : dissolution du précipité de fraction IV de plasma, élimination de la diatomite et des impuretés par centrifugation, et recueil du surnageant; addition de chlorure de sodium audit surnageant pour précipiter la protéine ApoA-I, et obtention du précipité d'ApoA-I par centrifugation; redissolution du précipité d'ApoA-I et sa filtration; séparation du filtrat de la procédure ci-dessus d'abord par chromatographie sur colonne anionique ensuite par chromatographie sur colonne hydrophobe pour obtenir une solution d'ApoA-I de pureté élevée.
PCT/CN2010/077463 2010-01-15 2010-09-29 Procédé de fabrication pour préparer apoa-i de pureté élevée à partir d'un précipité de fraction iv de plasma WO2011085598A1 (fr)

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CN201010022840XA CN102127165B (zh) 2010-01-15 2010-01-15 一种从血浆组分四沉淀中制备高纯ApoA-I的生产工艺
CN201010022840.X 2010-01-15

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Cited By (1)

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TWI508972B (zh) * 2011-03-04 2015-11-21 Kieu Hoang 製造個別的載脂蛋白、轉鐵蛋白及阿法１抗胰蛋白酶（ａ１ａｔ）或組合的轉鐵蛋白／載脂蛋白／人類白蛋白／ａ１ａｔ及所有新發現的蛋白質之第ｉｖ部分中發現的複合蛋白之製備及純化方法

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CN102977180B (zh) * 2012-11-06 2016-03-16 中国科学院过程工程研究所 一种综合利用Cohn组分IV的方法
JP6404333B2 (ja) * 2013-06-05 2018-10-10 シーエスエル、リミテッド アポリポタンパク質ａ−ｉ（アポａ−ｉ）の製造方法
CN104020301B (zh) * 2014-01-06 2015-11-18 宁波博泰生物技术有限公司 用于校准载脂蛋白a1和载脂蛋白b的校准物的制备方法
CN104513306B (zh) * 2014-12-15 2016-08-17 山西瑞亚力科技有限公司 载脂蛋白A1的纯化方法和ApoAI蛋白注射抗原
CN106279405A (zh) * 2016-09-23 2017-01-04 中国药科大学 一种Cohn组分四血浆功能蛋白提纯的方法
CN107033237B (zh) * 2017-05-11 2021-07-20 深圳市卫光生物制品股份有限公司 一种人血浆载脂蛋白a-i的分离纯化方法
CN108977423A (zh) * 2018-08-17 2018-12-11 集美大学 一种从猪肺中分离提纯血管紧张素转化酶的方法

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Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
TWI508972B (zh) * 2011-03-04 2015-11-21 Kieu Hoang 製造個別的載脂蛋白、轉鐵蛋白及阿法１抗胰蛋白酶（ａ１ａｔ）或組合的轉鐵蛋白／載脂蛋白／人類白蛋白／ａ１ａｔ及所有新發現的蛋白質之第ｉｖ部分中發現的複合蛋白之製備及純化方法

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