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High-capacity dewatering electric charge inducing color chromatogram medium and preparation method

A chromatographic medium and hydrophobic electricity technology, applied in the field of biochemical engineering, can solve the problems of low adsorption capacity and harsh elution conditions of hydrophobic charge-induced chromatography, and achieve the effect of ensuring activity, good stability and convenient regeneration

Inactive Publication Date: 2008-05-28
TIANJIN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Compared with traditional protein chromatography methods, hydrophobic charge induction chromatography still has the disadvantages of low adsorption capacity and harsher elution conditions than traditional chromatography methods.

Method used

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  • High-capacity dewatering electric charge inducing color chromatogram medium and preparation method
  • High-capacity dewatering electric charge inducing color chromatogram medium and preparation method
  • High-capacity dewatering electric charge inducing color chromatogram medium and preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Rinse the Sepharose CL-6B soaked in 20% ethanol with an average particle size of 87 μm with deionized water, and drain it with a water pump in an hour glass for 5 minutes; weigh 1 g of the drained Sepharose CL-6B and set Into a 50ml Erlenmeyer flask, add 1.5mL sodium hydroxide (0.8mol / L), 0.5mL epichlorohydrin, 2mL dimethyl sulfoxide, shake and react at 40°C and 170rpm for 3h. The epoxy-activated Sepharose CL-6B agarose gel medium can be obtained. After the activation medium was washed repeatedly in the hourglass to remove free epichlorohydrin, the epoxy modification density of the gel medium was determined to be 120 μmol / mL of the activation medium.

[0025] Weigh 1.0 g of the activation medium and place it in 5 mL of disodium hydrogen phosphate, which contains 0.6 mmol of histamine; place it in a constant temperature water bath at 60°C for 10 hours; After thoroughly rinsing with % ethanol and distilled water, add 0.5g / L sodium borohydride solution to reduce the epoxy...

Embodiment 2

[0027] The volumetric dosage of epichlorohydrin in embodiment 1 is 1.5mL, the volumetric dosage of 1.4mol / L sodium hydroxide is 5.0mL, other conditions are constant, and the epoxy group modification density in the obtained activation medium is 158.6 μ mol / mL activation medium. The concentration of 3-methylhistamine added to the coupling reaction was 2.0 mmol / L, and the coupling density of the ligand in the chromatographic medium was 114 μmol / mL.

Embodiment 3

[0029] Rinse the Sepharose CL-6B soaked in 20% ethanol with an average particle size of 87 μm with deionized water, and drain it with a water pump in an hour glass for 5 minutes; weigh 1 g of the drained Sepharose CL-6B and set Into a 50mL Erlenmeyer flask, add 1.5mL sodium hydroxide (0.5mol / L), 0.3mL epichlorohydrin, 2mL dimethyl sulfoxide, shake and react at 25°C and 170rpm for 3h. The epoxy-activated Sepharose CL-6B agarose gel medium can be obtained. After the activation medium was washed repeatedly in the hourglass to remove free epichlorohydrin, the epoxy modification density of the gel medium was determined to be 70 μmol / mL of the activation medium.

[0030] Weigh 1.0 g of the activation medium and place it in 5 mL of disodium hydrogen phosphate, the solution contains 0.5 mmol 4-aminomethylimidazole; place it in a constant temperature water bath at 60°C for 10 hours; , volume concentration 20% ethanol and distilled water, after thoroughly rinsing, add 0.5g / L sodium bor...

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Abstract

The invention discloses a preparation method of a hydrophobic charge induction chromatography medium with large capacity, and belongs to the protein chromatography separation technology in the field of biochemical engineering. The chromatography medium refers to the end of a space arm that is coupled with imidazol compounds as a chromatography matrix. The preparation method is that: the medium is activated after dimethyl sulfoxide, epichlorohydrin and sodium hydroxide solution are sequentially put into agarose gel medium; the activated medium is coupled with the imidazol compounds matrix in the alkaline dimethyl sulfoxide solution; finally, the epoxy left on the medium surface gets the needed chromatography medium after being reduced. The adsorption capacity of the chromatography medium to the protein achieves a moist medium of 80mg / ml and keeps relatively stable within a broader ionic strength scope from 0.1 mol / L to 1.0 mol / L; at the same time, the protein elutropic model which is finished by depending on the regulation of PH between 4.0 and 6.0 is more moderate, thereby having the wide application prospect in the separation and purification of biomacromolecule such as the protein, etc.

Description

technical field [0001] The invention relates to a large-capacity hydrophobic charge-induced chromatographic medium and a preparation method thereof, belonging to protein chromatographic separation technology in the field of biochemical engineering. Background technique [0002] In recent years, with the development of biotechnology, proteins closely related to human health have been mass-produced through gene recombination technology. The products obtained by mass-producing proteins with the help of genetically engineered bacteria contain not only target products, but also miscellaneous proteins produced by a large number of host cells during their own development and growth. At the same time, the widely used high-density fermentation technology achieves high yields of target products on the premise of providing more abundant carbon, nitrogen and inorganic salt ions. At the same time, the nutrients remaining in the fermentation broth also make the target product in a more c...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): B01J20/281C07K1/16
Inventor 史清洪沈芳芳孙彦白姝董晓燕
Owner TIANJIN UNIV
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