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Method for purifying recombinant protein

A technology of recombinant protein and protein, applied in the field of protein chemistry, can solve the problems of long process route, complicated operation, narrow application range, etc., and achieve the effect of improving purification efficiency, low protein adsorption amount, and wide application range

Active Publication Date: 2021-07-16
LUNAN PHARMA GROUP CORPORATION
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0014] In view of the shortcomings of the recombinant protein purification method in the prior art, such as narrow application range, long process route, complicated operation, high production cost, and inability to realize large-scale industrial production of recombinant protein, this application provides a purification method with a wide application range and a process route Short and promising method for industrial production of recombinant proteins with high purity

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  • Method for purifying recombinant protein
  • Method for purifying recombinant protein

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Experimental program
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Effect test

Embodiment 1

[0066] A. Ultrafiltration: The primary purification sample (103.4L, 0.3g / L) is clarified and filtered through a cellulose membrane PLCHK-C 100KD tangential flow membrane filter. The solution and the buffer were replaced at a volume ratio of 1:4, and the combined filtrate was the filtered sample of leukocyte inhibitory factor and hirudin chimeric protein, which was collected and stored at 2-8°C, with a yield of 93.6%;

[0067] B. Hydrophobic chromatography: At room temperature, use Phenyl Sepharose High Performance column with a diameter of 10cm and a height of 20cm (column bed volume 1.57L), and then use 5mM Tris-HCl and 300mM ammonium sulfate buffer solution with a pH of 7.5 After equilibration, load the filtered sample containing leukocyte inhibitory factor and hirudin chimeric protein obtained in step A. After loading the sample, wash the chromatography column with 5mM Tris-HCl and 300mM ammonium sulfate buffer solution with a pH of 7.5 until the effluent is detected. The v...

Embodiment 2

[0072] A. Ultrafiltration: The primary purification sample (107.7L, 0.3g / L) was clarified and filtered through a polyethersulfone membrane BioMax 100KD tangential flow membrane filter. After being concentrated 20 times, the concentrated protein solution was purified by ultrafiltration membrane bag Replace with buffer solution at a volume ratio of 1:5, and combine the filtrate, which is the filtered sample of leukocyte inhibitory factor and hirudin chimeric protein, collected and stored in an environment of 2-8°C, with a yield of 92.6%;

[0073] B. Hydrophobic chromatography: At room temperature, use Phenyl Sepharose Fast Flow to pack the column with a diameter of 10cm and a height of 20cm (column bed volume 1.57L), and then use 5mM Tris-HCl and 300mM ammonium sulfate buffer solution with a pH of 7.0 After equilibration, load the filtered sample containing leukocyte inhibitory factor and hirudin chimeric protein obtained in step A. After loading the sample, wash the chromatograp...

Embodiment 3

[0078] A. Ultrafiltration: The primary purification sample (99.5L, 0.3g / L) is clarified and filtered through a cellulose membrane PLCHK-C 200KD tangential flow membrane filter. The solution and the buffer were replaced at a volume ratio of 1:3, and the combined filtrate was the filtered sample of leukocyte inhibitory factor and hirudin chimeric protein, which was collected and stored at 2-8°C, with a yield of 93.4%;

[0079] B. Hydrophobic chromatography: At room temperature, use Phenyl Sepharose High Performance column with a diameter of 10cm and a height of 20cm (column bed volume 1.57L), and then use 5mM Tris-HCl and 300mM ammonium sulfate buffer with a pH of 8.0 After equilibration, load the filtered sample containing leukocyte inhibitory factor and hirudin chimeric protein obtained in step A. After loading the sample, wash the chromatography column with 5mM Tris-HCl and 300mM ammonium sulfate buffer solution with pH 8.0 until the effluent is detected. The value is the bas...

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Abstract

The invention belongs to the field of protein chemistry, and particularly relates to a method for purifying recombinant protein. The method comprises the following steps of clarifying and concentrating fermentation liquor containing target recombinant protein through at least one tangential flow membrane filtration step, and then carrying out hydrophobic chromatography, reversed-phase chromatography, pyrogen removal, ultrafiltration and bottling, and obtaining the recombinant leukocyte inhibiting factor and hirudin chimeric protein with the purity of 99.5%. The method is suitable for large-scale industrial production of the recombinant leukocyte inhibiting factor and hirudin chimeric protein.

Description

technical field [0001] The invention belongs to the field of protein chemistry, and in particular relates to a method for purifying recombinant protein. Background technique [0002] Recombinant protein drugs are usually expressed in Escherichia coli, yeast, and mammalian cells, among which large-scale cultivation and production of protein drugs combined with genetic engineering has become the mainstream in the field of biopharmaceuticals today. Many clinical protein drugs require higher doses or long-term medication, and the market demand is large. Therefore, the large-scale production of this type of biopharmaceuticals requires high process quality and must meet strict pharmaceutical standards. Antibody or Fc fusion protein drugs can usually achieve more than 90% purity by one-step Protein A affinity chromatography, and other contaminating impurities such as shed Protein A, host cell protein (HCP), aggregates (D / A) and DNA etc. are mainly removed by chromatography methods...

Claims

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Application Information

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IPC IPC(8): C07K1/36C07K1/34C07K1/20C07K1/18C07K19/00
CPCC07K1/36C07K1/34C07K1/20C07K1/18C07K14/5415C07K14/815C07K2319/00
Inventor 朱梅梅柳常青刘忠
Owner LUNAN PHARMA GROUP CORPORATION
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