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A high-capacity agarose chromatography medium and its preparation method

A chromatography medium and agarose technology, which is applied in the field of high-capacity agarose chromatography medium and its preparation, can solve problems such as preventing proteins from entering the medium, and achieve increased medium load, increased number of sites, and protein loading. The effect of volume increase

Active Publication Date: 2017-12-22
SENHUI MICROSPHERE TECH SUZHOU CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

However, we have also found that the dextran introduced by this method is homogeneously distributed in the entire medium, and the whole grafting process increases the binding sites between the medium and the protein to improve its loading, and also affects the medium to a certain extent. The internal pores are blocked, which prevents the protein from entering the medium, and has an adverse effect on the further increase of the medium capacity (such as literature 5: Zhang Jingfei, Zhao Lan, Huang Yongdong, Zhang Zhigang, Ma Guanghui, Su Zhiguo. Dextran grafted type Preparation and performance of high-capacity metal chelating media. Chinese Journal of Process Engineering [J], 2015,15(1):111-118)

Method used

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  • A high-capacity agarose chromatography medium and its preparation method
  • A high-capacity agarose chromatography medium and its preparation method

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Experimental program
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Effect test

Embodiment 1

[0033] Primary activation: Accurately weigh 10g of Sepharose 6FF (6% agarose solution, crosslinking matrix), add it to a three-hole kettle, add 0.01g sodium borohydride, 1g anhydrous sodium sulfate, 2mL 30% NaOH solution, 1mL allyl Glycidyl ether and 3mL water were reacted at 50°C for 18h. After the reaction stopped, the medium was repeatedly washed with a large amount of deionized water. Prepare an allyl-activated medium with an allyl density of 150 μmol / g to dry the wet glue.

[0034] Primary bromination: add 20 mL of water, 1.2 g of sodium acetate, and 2 mL of bromine water to 10 g of allyl activation medium, and react for 1 h at room temperature. After the reaction, the medium was washed with plenty of water. Obtain a partially brominated agarose medium with an allyl density of 105 μmol / g and drain the wet gel.

[0035] Graft dextran: Take 10g of partially brominated agarose medium, add 15mL 0.6g / ml dextran solution (dextran T40, molecular weight 40kDa), 7mL water, 15mL...

Embodiment 2

[0042] Primary activation: Accurately weigh 10 g of cross-linked white spheres prepared with a concentration of 2% agarose solution, and the rest are the same as in Example 1. Prepare an allyl-activated medium with an allyl density of 10 μmol / g to dry the wet glue.

[0043] Primary bromination: Add 10 g of allyl-activated medium to 20 mL of water, 1.2 g of sodium acetate, and 1 mL of bromine water, and react for 1 h at room temperature. After the reaction, the medium was washed with plenty of water. Obtain a partially brominated agarose medium with an allyl density of 1 μmol / g and drain the wet gel.

[0044] Graft dextran: Take 10g of partially brominated agarose medium, add 15mL 0.6g / ml dextran solution (dextran T2, molecular weight 2kDa), 7mL water, 10mL 1M NaOH solution (3.6mg sodium borohydride), React at 30°C for 18 hours, and wash the medium with a large amount of deionized water after the reaction. Prepare a dextran-grafted medium with a dextran content of 0.1 mg / mL ...

Embodiment 3

[0051] One-time activation: Accurately weigh 10g of cross-linked white balls prepared by 15% agarose solution concentration, use allyl bromide as the activator, add 0.01g sodium borohydride, 1g anhydrous sodium sulfate, 2mL 30% NaOH solution, 3mL Allyl glycidyl ether and 3mL water were reacted at 50°C for 12h. An allyl-activated medium was prepared with an allyl density of 2000 μmol / g to dry the wet glue.

[0052] Primary bromination: add 20 mL of water to 10 g of allyl activation medium, 1.2 g of sodium acetate, and 3 mL of bromine water, and react for 1 h at room temperature. After the reaction, the medium was washed with plenty of water. Obtain a partially brominated agarose medium with an allyl density of 1900 μmol / g and drain the wet gel.

[0053] Graft dextran: Take 10g of partially brominated agarose medium, add 15mL 0.6g / ml dextran solution (dextran T150, molecular weight 150kDa), 7mL water, 20mL 1M NaOH solution (3.6mg sodium borohydride), React at 30°C for 18 hour...

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Abstract

The present invention relates to the present invention provides a high-capacity agarose chromatography medium, which is characterized in that the medium is composed of a shell and an inner core, wherein the shell part is covalently bonded with linear macromolecules, such as dextran, etc. Both the shell part and the core part are coupled with ligands, and the types of ligands include metal ions, hydrophobic ligands, ion exchange groups and proteins, etc. The invention also includes a method for preparing the high-capacity agarose chromatographic medium. Taking the metal chelation chromatography medium as an example, compared with the conventional medium, the loading capacity of the medium prepared by this method for His-tagged recombinant protein is about 2.5 times that of the conventional medium. This high-capacity agarose chromatographic medium has good application prospects in the field of biological separation and purification.

Description

technical field [0001] The invention belongs to the field of biotechnology, and in particular relates to a high-capacity agarose chromatographic medium, a preparation method thereof, and an application in the field of purification of protein and other biomacromolecules. Background technique [0002] Protein separation and purification is one of the key factors in the field of protein science and product engineering. In the field of proteins and related biologically active substances with complex sources, different structures and properties, how to achieve efficient separation and purification is a problem that researchers have been working hard to solve. Chromatography medium is the most basic component of chromatography technology, and its structure and properties are one of the most critical factors affecting the final separation and purification of proteins. With the continuous development of genetic engineering proteology, protein engineering, vaccine engineering and oth...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): B01J20/24B01J20/26B01J20/30
Inventor 赵岚黄永东巩方玲马光辉苏志国
Owner SENHUI MICROSPHERE TECH SUZHOU CO LTD
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