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Multidimensional liquid phase chromatography separation system and separation method for protein separation

A multi-dimensional liquid chromatography and separation system technology, applied in material separation, analytical materials, instruments, etc., can solve problems such as no technical solutions and technical parameters, no way to scale up the preparation scale, no strong theoretical support, etc.

Active Publication Date: 2013-04-24
XIAN AOLAN SCI & TECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Yet this method also has the following problems: (1) because of being limited by the maximum flow rate of the chromatograph (only 5 milliliters / minute) and the maximum volume of each collecting tube in the collecting device (only 5 milliliters), its It can only be carried out on the analysis scale, and cannot be scaled up to the preparation scale, let alone the production scale; (2) Because the instrument only uses one chromatographic column to separate proteins, it is impossible to implement the mD- The purpose of high purity purified by LC method (for example, the purity of injected insulin requires protein > 99%); (3) because there is no strong theoretical support, it can only be operated manually by experience, and it is impossible to design a program and control it with computing software. And this is a necessary condition of today's modern instruments, and does not include peptides in the scope of application; (4) The chromatographic column used is a specially prepared "2D-LC", and there is no commercial product, which is difficult for others to implement; and ( 5) More importantly, all that supports the implementation of this method are only accessories of several kinds of equipment. The disorderly patchwork on the existing chromatograph has neither a framework for the overall idea of ​​manufacturing a new equipment, nor any possibility. Technical solutions and technical parameters for implementation

Method used

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  • Multidimensional liquid phase chromatography separation system and separation method for protein separation
  • Multidimensional liquid phase chromatography separation system and separation method for protein separation
  • Multidimensional liquid phase chromatography separation system and separation method for protein separation

Examples

Experimental program
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Effect test

Embodiment 1

[0324] Example 1 2D-LC (WCX×HIC) separation of 7 proteins by commercial column Olan WCX column

[0325] According to the aforesaid two-dimensional liquid chromatography separation system and two-dimensional liquid chromatography separation method, this example contains myoglobin (Myo), ribonuclease (RNase), cytochrome c (Cyt-c), α-chymosin Proprotein (α-Chy), lysozyme (Lys), carbonic anhydrase (Car) and α-amylase (α-Amy) were completely isolated from this mixture of 7 proteins. Among them, the first-dimensional separation adopts the weak cation exchange (WCX) separation mode, while the second-dimensional separation adopts the hydrophobic interaction (HIC) separation mode. The chromatographic column used in this example is a commercial Shim-pack PA-CM WCX chromatographic column, which is actually a WCX / HIC mixed mode chromatographic column (150 mm × 7.9 mm), in the first dimension separation of WCX mode and HIC mode This column is used in all second dimension separations.

[...

Embodiment 2

[0346] Example 2 Three-dimensional (multi-dimensional) 3D-LC (WCX×HIC×SEC) for α-chytrogenin (α-Chy) and pancreatic Protease (Try) is simultaneously purified and desalted. G4000SWXL

[0347] According to the aforementioned 2D-LC separation system and 2D-LC separation method, in this example, α-Chy and Try were simultaneously purified from the bovine pancreas extract prepared by conventional methods and degreased, and then the separated proteins were separated desalination. Among them, the first dimension separation adopts WCX mode, and the second dimension separation adopts HIC mode. Due to the large volume of the two-dimensional effluent and the concentration of the target protein in the effluent is constantly changing with time, and it cannot be completely injected into the SEC column at one time, it must be collected in an oval storage tube and accompanied by After stirring by the oscillator to make it evenly mixed, the samples were injected into the third dimension in S...

Embodiment 3

[0368] Example 3, On-line single-column two-dimensional liquid chromatography column (2D (WCX, HIC) rapid purification of cytochrome c in bovine pancreas

[0369] Firstly, cytochrome c is crudely extracted from bovine pancreas: the fresh bovine pancreas is removed from oil and connective tissue and washed, and then quickly stored in a -20°C refrigerator; after the frozen bovine pancreas is sliced, use a meat grinder Grind into meat powder, then add 2 times the volume of sulfuric acid acidified (pH=4) water to extract under stirring conditions, and then use 1mo / L H 2 SO 4 Adjust the pH once to stabilize the pH between 3.5-4.0, and extract with stirring at low temperature (4°C) for more than 12 hours; use 1.0mol / L NH 4 Adjust the pH to 6.5 with OH, press and filter with four layers of gauze, collect the filtrate, and let it stand at 4°C; finally, centrifuge at 10,000rpm for 20min, and collect the supernatant as the crude extract of cytochrome c. All operations should be carrie...

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Abstract

The invention relates to a multidimensional liquid phase chromatography separation system and separation method for protein separation. The multidimensional liquid phase chromatography separation system for protein separation comprises a mobile phase storage tank, a first liquid delivery device, a second liquid delivery device, a first sampling device, a second sampling device, a separation device, a collection and storage device, at least two liquid discharge devices and a flow path switch. When implementing every dimension of liquid phase chromatography separation, the collection and storage device collects a target intermediate fraction for the next liquid phase chromatography separation step and stores the target intermediate fraction in the collection and storage unit; and in the second or more dimension liquid phase chromatography separation process, all the target intermediate fraction from the collection and storage unit is mixed with the mobile phase through a sampling mixer of the second sampling device, and the first liquid delivery device and the second liquid delivery device are utilized to adjust and meter the quantity of the delivered mobile phase, so that the concentration of the eluting agent in the sampled mixed solution is lower than the concentration of the critical migration eluting agent for all the target proteins to be preserved in the separation process in the target intermediate fraction.

Description

technical field [0001] The invention relates to a multidimensional liquid chromatography separation system for protein separation, in particular to a multidimensional liquid chromatography separation system for online fast separation of proteins. The invention also relates to a separation method for protein separation using the liquid chromatography separation system. Background technique [0002] Liquid chromatography is the most effective method for analyzing, separating and purifying proteins. It is widely used in the extraction and separation of active proteins from natural animal and plant body fluids, and the preparation and production of various recombinant protein drugs produced by genetic engineering. It is also a biochemical laboratory Indispensable equipment, e.g. in protein and peptide separations for the search for "marker" proteins in proteomics research. Because the environmental components of such biomacromolecules are very complex, it is usually called the ...

Claims

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Application Information

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IPC IPC(8): G01N30/02G01N30/10
Inventor 耿信笃
Owner XIAN AOLAN SCI & TECH
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