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Separation and purification method of recombinant protein

A recombinant protein, separation and purification technology, applied in the field of protein chemistry, can solve the problems of long process route, high production cost, high aggregate content, etc., and achieve the effects of improving purification efficiency, simplifying operation, and reducing protein adsorption

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

AI Technical Summary

Problems solved by technology

[0014] In view of the deficiencies in the prior art such as inability to effectively remove endotoxin, high polymer content, long process route, complicated operation, high production cost, and inability to realize large-scale industrial production of recombinant proteins, the present application provides a method that can effectively remove endotoxin, A method with reduced aggregate content, short process route, and promising industrial production of recombinant proteins with high purity

Method used

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  • Separation and purification method of recombinant protein
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Examples

Experimental program
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Effect test

Embodiment 1

[0071] A. Ultrafiltration: The primary purified sample (120.5L, 0.25g / L) containing insulin glargine precursor was clarified and filtered through cellulose membrane regenerated cellulose P2C-100-C01 100KD tangential flow membrane filtration, concentrated 30 times Finally, replace the concentrated protein solution and 0.1M ammonium sulfate buffer solution at a volume ratio of 1:4 with an ultrafiltration membrane bag for at least 3 times, and combine the filtrate, which is the filtered sample of insulin glargine precursor, which is collected and stored in 2-8 In ℃ environment, the yield is 95.2%;

[0072] B. Digestion: Use bovine trypsin to digest the filtered sample containing insulin glargine precursor obtained in step A, add 35.85 mg of bovine trypsin, adjust the pH to 9.0, control the temperature at 4°C, digest for 12 hours, and adjust the pH to 3 .0 to terminate the enzyme digestion, and obtain the enzyme digestion solution, which is stored in an environment of 2-8°C, and t...

Embodiment 2

[0078] A. Ultrafiltration: The primary purification sample (120.9L, 0.25g / L) containing insulin precursor was clarified and filtered by PLC-100-C01200KD tangential flow membrane filtration, concentrated 20 times, and concentrated by ultrafiltration membrane bag The protein solution and 0.1M ammonium sulfate buffer were replaced at least 4 times at a volume ratio of 1:3, and the combined filtrate was the filtered sample of insulin glargine precursor, which was collected and stored in an environment of 2-8°C, with a yield of 94.8%;

[0079] B. Digestion: Use bovine trypsin to digest the filtered sample obtained in step A, add 40.94 mg of bovine trypsin, adjust the pH to 9.5, control the temperature at 8°C, digest for 10 hours, adjust the pH to 3.0 to stop the digestion, and obtain Enzyme cutting solution, the enzyme cutting solution is stored in an environment of 2-8°C, and the yield is 60.1%;

[0080] C. Reverse-phase chromatography: Use Source 30RPC to pack a column with a col...

Embodiment 3

[0085] A. Ultrafiltration: The primary purification sample (120.4L, 0.25g / L) containing the insulin glargine precursor was clarified and filtered through polyethersulfone P2B-100-A05 300KD, tangential flow membrane filtration, concentrated 50 times, Replace the concentrated protein solution and 0.1M ammonium sulfate buffer at a volume ratio of 1:5 for at least 3 times with an ultrafiltration membrane bag, combine the filtrates, collect and store them in an environment at 2-8°C, with a yield of 94.8%;

[0086] B. Digestion: Use porcine trypsin to digest the filtered sample obtained in step B, add 31.70 mg of porcine trypsin, adjust the pH to 8.0, control the temperature at 6°C, digest for 13 hours, adjust the pH to 3.0 to stop the digestion, and obtain Enzyme cutting solution, the enzyme cutting solution is stored in an environment of 2-8°C, and the yield is 60.3%;

[0087]C. Reversed-phase chromatography: use Source 30RPC to pack a column with a column diameter of 10cm and a h...

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Abstract

The invention belongs to the field of protein chemistry, and particularly relates to a separation and purification method of recombinant protein. The separation and purification method of recombinant protein comprises the following steps: centrifuging fermentation broth containing target recombinant protein to obtain wet thalli; carrying out primary purification treatment such as bacterium breaking, washing and renaturation; carrying out filtering through at least one tangential flow membrane, and carrying out enzyme digestion; and then, carrying out at least two times of reversed phase chromatography, depyrogenation, ultrafiltration, bottling and the like to obtain insulin glargine with the purity of 99.7%. The method can effectively remove endotoxin and reduce impurities such as polymers, and is suitable for large-scale industrial production of insulin glargine.

Description

technical field [0001] The invention belongs to the field of protein chemistry, and in particular relates to a method for separating and purifying recombinant proteins. Background technique [0002] Recombinant protein is a protein obtained in Escherichia coli, yeast, insect cells, mammalian cells and other systems by using recombinant DNA or RNA technology. It has the advantages of significant curative effect, strong specificity, low toxicity and small side effects. At present, most products in the field of biomedicine are recombinant protein products, such as enzymes, recombinant cytokines, Fc fusion proteins, chimeric proteins, monoclonal antibodies and their derivatives, etc. [0003] The Escherichia coli expression system is currently the most commonly used exogenous protein expression system. It has the advantages of short cycle, low culture cost, mature metabolic regulation, and convenient operation. It has become the preferred expression system for recombinant protei...

Claims

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Application Information

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IPC IPC(8): C07K1/36C07K1/34C07K1/20C12N15/70
CPCC07K1/36C07K1/34C07K1/20C12N15/70
Inventor 张贵民朱梅梅柳常青刘忠
Owner LUNAN PHARMA GROUP CORPORATION
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