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Method for purifying hyaluronidase by affinity membrane chromatography

A hyaluronidase and affinity membrane technology is applied in the field of affinity membrane chromatography to purify hyaluronidase, and achieves the effect of solving the needs of medical drugs, good economic benefits and social benefits

Inactive Publication Date: 2019-06-25
青岛冠龙生物制药有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Chinese patent 201310643506.X "A Method for Extracting Hyaluronidase Quality" only uses ordinary chromatographic separation to purify hyaluronidase. This patent uses affinity membrane chromatography to purify hyaluronidase, which has a higher purification factor. It is the first of its kind, and there is no relevant introduction on the purification of hyaluronidase by affinity membrane chromatography in domestic related papers, patents and other literature

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] 1.1 Activation of nylon membrane

[0025] Nylon membranes were first hydrolyzed in 1mol / L HCL at 40°C for 4 hours, and then activated with formaldehyde. Then 10 pieces of hydrolyzed nylon membranes were immersed in 20mL of formaldehyde solution (>36.5%), and 0.2ml of 85% phosphoric acid was added. ℃ reaction for 7 hours, 40-50 ℃ hot water wash 6 times

[0026] 1.2 Chitosan modification of nylon membrane

[0027] Immerse the above-mentioned formaldehyde-activated nylon membrane in 10ml of chitosan solution with a mass fraction of 1.5% (dissolved in 1% acetic acid by volume fraction), react at room temperature for 1h, then transfer to an oven at 80°C, take it out after 1h, and wash with 1% Acetic acid and deionized water were used to wash away unreacted chitosan.

[0028] 1.3 Preparation of dye affinity membrane

[0029] 20 sheets of the above-mentioned modified nylon membranes were immersed in 200ml of 10mg / ml Cibacron Blue F3GA aqueous solution, sealed and stirred at...

Embodiment 2

[0035] 1.1 Activation of nylon membrane

[0036] Nylon membranes were first hydrolyzed in 1mol / L HCL at 40°C for 4 hours, and then activated with formaldehyde. Then, 10 pieces of hydrolyzed nylon membranes were immersed in 20mL formaldehyde solution (>36.5%), and 0.2ml 85% phosphoric acid was added, at 60 ℃ reaction for 7 hours, 40-50 ℃ hot water wash 6 times

[0037] 1.2 Chitosan modification of nylon membrane

[0038] Immerse the above-mentioned formaldehyde-activated nylon membrane in 10ml chitosan solution with a mass fraction of 1.5% (dissolved in 1% acetic acid by volume fraction), react at room temperature for 1h, then transfer to an oven at 80°C, take it out after 1h, and wash with 1% Acetic acid and deionized water were used to wash away unreacted chitosan.

[0039] 1.3 Preparation of dye affinity membrane

[0040] 20 sheets of the above-mentioned modified nylon membranes were immersed in 200ml of 10mg / ml Cibacron Blue F3GA aqueous solution, sealed and stirred at 6...

Embodiment 3

[0046] 1.1 Activation of nylon membrane

[0047] Nylon membranes were first hydrolyzed in 1mol / L HCL at 40°C for 4 hours, and then activated with formaldehyde. Then, 10 pieces of hydrolyzed nylon membranes were immersed in 20mL formaldehyde solution (>36.5%), and 0.2ml 85% phosphoric acid was added, at 60 ℃ reaction for 7 hours, 40-50 ℃ hot water wash 6 times

[0048] 1.2 Chitosan modification of nylon membrane

[0049] Immerse the above-mentioned formaldehyde-activated nylon membrane in 10ml chitosan solution with a mass fraction of 1.5% (dissolved in 1% acetic acid by volume fraction), react at room temperature for 1h, then transfer to an oven at 80°C, take it out after 1h, and wash with 1% Acetic acid and deionized water were used to wash away unreacted chitosan.

[0050] 1.3 Preparation of dye affinity membrane

[0051] 20 sheets of the above-mentioned modified nylon membranes were immersed in 200ml of 10mg / ml Cibacron Blue F3GA aqueous solution, sealed and stirred at 6...

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PUM

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Abstract

The invention provides a method for purifying a hyaluronidase by an affinity membrane chromatography, which uses a nylon membrane as a matrix, a method of bonding chitosan is used to modify the nylonmembrane, so that the non-specific adsorption of the membrane is greatly reduced, and a novel dye affinity membrane is obtained, which has good adsorption to hyaluronidase and has a high purificationratio.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for purifying hyaluronidase by affinity membrane chromatography. Background technique [0002] Hyaluronidase, also known as hyaluronidase, is abundant in the testes of mammals. It can hydrolyze hyaluronic acid and significantly reduce its viscosity, which is beneficial for sperm to enter the egg during fertilization. It also exists in sperm, submandibular gland, bee venom, snake venom, skin, spleen, leech and lysosome of cells. [0003] The stability of hyaluronidase is good, and the activity will not be lost when heated at 42°C for 60 minutes; 80% of the activity can be retained when heated at 100°C for 5 minutes. Cooling after heat inactivation can restore some vitality. The enzyme is still relatively stable at pH below 5 or above pH 8. [0004] Medicinal hyaluronidase is white or yellowish powder, odorless, soluble in water, insoluble in acetone, ether, ethanol and oth...

Claims

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Application Information

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IPC IPC(8): C12N9/26
Inventor 刘乃山夏衬来宋超龙
Owner 青岛冠龙生物制药有限公司
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