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Separation method of consensus interferon Alpha (cIFN) isomer

A technology of alpha interferon and separation method, applied in the field of protein separation and purification

Active Publication Date: 2012-08-22
EAST CHINA UNIV OF SCI & TECH +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] Nowadays, some achievements have been made in the separation of macromolecular active substances by using HSCCC, such as the separation of four standard proteins, cytochrome c, myoglobin, ovalbumin and bovine hemoglobin, and the separation of chicken albumin, but the application of HSCCC in the separation of molecular weight is only cIFN isomers with a difference of 4 protons have not been reported at home and abroad

Method used

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  • Separation method of consensus interferon Alpha (cIFN) isomer
  • Separation method of consensus interferon Alpha (cIFN) isomer
  • Separation method of consensus interferon Alpha (cIFN) isomer

Examples

Experimental program
Comparison scheme
Effect test

preparation example Construction

[0056] Preparation of isolated samples

[0057] Weigh a certain amount of PEG and phase-forming salt, add a certain amount of cIFN isomer sample to dissolve so that the composition is the same as the separation system used (the sample itself is a solution, calculate the ratio, just add PEG and salt directly), stir slightly Allow all solids to dissolve completely.

[0058] Determination of partition coefficient

[0059] HPLC area division method

[0060] Stationary phase: C4 column (Grace Vydac company)

[0061] Mobile phase: Phase A: water + 0.01% trifluoroacetic acid (TFA), phase B: 60% acetonitrile + 30% dioxane + 10% water + 0.01% TFA;

[0062] Gradient elution conditions: Phase B concentration 0-5 minutes, 5%; 5-55 minutes, 5%-60%; 55-60 minutes, 60%.

[0063] Determination of Retention Rate of Upper Phase (also known as Stationary Phase) in Aqueous Two-Phase System

[0064] Fill the HSCCC separation column with the upper phase (stationary phase) in the two-phase syst...

Embodiment 1

[0068] Separation of cIFN Isomers by High Speed ​​Countercurrent Chromatography Two-Phase System

[0069] Materials and methods

[0070] TBE-200V high-speed countercurrent chromatography was purchased from Shanghai Tongtian Biotechnology Co., Ltd.

[0071] The cIFN isomer is obtained by subjecting cIFN fermentation supernatant to ion exchange chromatography and then hydrophobic chromatography. Two bands appear on the 19KD position of the SDS-PAGE band, and two peaks appear on the HPLC, (see figure 1 ), but mass spectrometry showed the same substance.

[0072] The sources of PEG2000, PEG4000, trisodium citrate, and citric acid are as follows:

[0073] name

source

Polyethylene glycol (PEG)

Shanghai Chemical Reagent Company

Citric Acid, Sodium Citrate, Dipotassium Hydrogen Phosphate, Potassium Dihydrogen Phosphate

Shanghai Chemical Reagent Company

Acetonitrile, dioxane, methanol, isopropanol, trifluoroacetic acid, etc.

Sinopharm Ch...

Embodiment 2

[0079] Separation of cIFN Isomers by High Speed ​​Countercurrent Chromatography Two-Phase System

[0080] Materials and methods

[0081] TBE-200V high-speed countercurrent chromatography was purchased from Shanghai Tongtian Biotechnology Co., Ltd.

[0082] The cIFN isomer is prepared by subjecting cIFN fermentation supernatant to ion exchange chromatography and then hydrophobic chromatography. There are two bands on SDS-PAGE, and two peaks on HPLC, (see figure 1 ), but mass spectrometry showed the same substance.

[0083]PEG2000, PEG4000, trisodium citrate, citric acid, their sources are as described in Example 1.

[0084] A TBE-200V high-speed countercurrent instrument is used, equipped with a constant flow pump, a 15ml injection valve, a polytetrafluoroethylene column with a column volume of 200ml, and a UV ultraviolet detector. Weigh 12g of PEG2000, 96g of PEG4000, 108g of trisodium citrate, 12g of citric acid according to the mass ratio of 1:8:9:1, add 972ml of pure wa...

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Abstract

The invention discloses a method for separating consensus interferon Alpha (cIFN) isomer by high-speed counter current chromatography. The condition of the high-speed counter current chromatography isas follows: 5-26 percent (w / w) of polyethylene glycol, 7-28 percent (w / w) inorganic or organic acid or salt thereof are mixed with the balance of water, a double water phase system is obtained by thesettling, the upper phase is a fixed phase, the lower phase is a mobile phase, and the weight percent uses the total weight of the double water phase system as the reference; the rotating speed of ahigh-speed counter current chromatography main machine is 600-1200t / min; and the flow rate of the mobile phase is 0.5-1.5ml / min.

Description

technical field [0001] The present invention relates to the separation and purification of proteins, in particular to a separation method of recombinant human compound alpha interferon (cIFN) isomers. Background technique [0002] Recombinant human composite alpha interferon (cIFN) is a non-naturally occurring type I alpha interferon with a recombinant amino acid sequence. Concentrating the advantages of various natural interferons, it has a stronger and broader antiviral effect. In vitro antiviral activity experiments have shown that the antiviral activity of cIFN is 5-10 times that of the other two commonly used interferons (IFN-α2a, IFN-α2b). The total cure rate of compound alpha interferon in the treatment of hepatitis C is as high as 40%, and it shows a high degree of antiviral activity against SARS. [0003] The complex interferon-α monomer protein with the correct conformation contains two pairs of intramolecular disulfide bonds, but in the process of cIFN preparati...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K1/16
Inventor 庄英萍杨琪郝玉有储炬程杰王维娜符晓辉张嗣良王永红
Owner EAST CHINA UNIV OF SCI & TECH