Ulinastatin purification method based on hydrophobic interaction column

A technology of ulinastatin and hydrophobic column, which is applied in the field of medicine, can solve the problems of low yield, low purity and instability of ulinastatin, and achieve high yield, huge economic and social benefits, and stability Good results

Inactive Publication Date: 2016-07-13
GUANGDONG TECHPOOL BIO-PHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] The purpose of the present invention is to overcome the defects of the existing ulinastatin that the purity is not high, the yield is too low, and instability, and adopt the method of purifying ulinastatin based on hydrophobic column chromatography, thereby improving the yield and purity of ulinastatin

Method used

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  • Ulinastatin purification method based on hydrophobic interaction column

Examples

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Comparison scheme
Effect test

Embodiment 1

[0028] Ulinastatin crude product purification steps:

[0029] 1) Pretreatment: Dissolve 100g of crude ulinastatin in 200ml of water, filter with diatomaceous earth, collect the filtrate, add ZnCl 2 Make its final concentration 150mmol / L, precipitate with precooled 95% ethanol according to the volume ratio of sample solution: 95% ethanol as 1: 1.1 (ml / ml), stir for 30min, plate and frame filter, get the supernatant, Adjust conductance to 55ms / cm, adjust pH to 4.0 with glacial acetic acid. The volume of the sample solution: 95% ethanol is 1:2.3, stirred for 30 minutes, filtered with a plate and frame, and the precipitate is taken; the precipitate is dissolved in 3 times the volume of water.

[0030] 2) Loading and washing: use pH 4.0, 1.5mol / L (NH 4 ) 2 SO 4 - 0.15mol / L acetate buffer equilibrates the chromatography column PhenylBio-sepHP, loads the sample, the protein concentration of the sample solution is about 30mg / mL gel, use pH4.0, 1.5mol / L (NH 4 ) 2 SO 4 - Wash wit...

Embodiment 2

[0033] Ulinastatin crude product purification steps:

[0034] 1) Pretreatment: Dissolve 100g of crude ulinastatin in 200ml of water, filter with diatomaceous earth, collect the filtrate, add ZnCl 2Make its final concentration 150mmol / L, precipitate with precooled 95% ethanol by the volume ratio of sample solution: 95% ethanol as 1: 1.1 (ml / ml), stir for 30min, centrifuge at 5000r / min for 20min, take the supernatant solution, adjust the conductance to 55ms / cm, and adjust the pH to 3.5 with glacial acetic acid. The volume of the sample solution: 95% ethanol is 1:2.3, stirred for 30 minutes, filtered with a plate and frame, and the precipitate is taken; the precipitate is dissolved in 3 times the volume of water.

[0035] 2) Loading and washing: use pH 3.6, 1.5mol / L (NH 4 ) 2 SO 4 - 0.1mol / L acetate buffer equilibrates the ButylBio-sepHP chromatography column, loads the sample, the protein concentration of the sample solution is about 30mg / mL gel, and uses pH3.6, 1.5mol / L (NH...

Embodiment 3

[0038] Ulinastatin crude product purification steps:

[0039] 1) Pretreatment: Dissolve 100g of crude ulinastatin in 200ml of water, filter with diatomaceous earth, collect the filtrate, add ZnCl 2 Make its final concentration 150mmol / L, precipitate with precooled 95% ethanol by the volume ratio of sample solution: 95% ethanol as 1: 1.1 (ml / ml), stir for 30min, centrifuge at 5000r / min for 20min, take the supernatant solution, adjust the conductance to 55ms / cm, and adjust the pH to 4.5 with glacial acetic acid. The volume of the sample solution: 95% ethanol is 1:2.3, stirred for 30 minutes, filtered with a plate and frame, and the precipitate is taken; the precipitate is dissolved in 3 times the volume of water.

[0040] 2) Loading and washing: use pH 3.5, 1.5mol / L (NH 4 ) 2 SO 4 -0.1mol / L acetate buffer equilibrates the chromatography column PhenylBio-sepFF, loads the sample, the protein concentration of the sample solution is about 30mg / mL gel, and uses pH 4.5, 1.5mol / L (...

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Abstract

The invention belongs to the technical field of medicine, and particularly relates to a ulinastatin purification method based on a hydrophobic interaction column.The method specifically comprises the steps that pretreatment such as deposition, filtration and pH regulation is performed on crude ulinastatin, separation and purification are performed on the ulinastatin through the hydrophobic interaction column, impure protein and colored groups are removed, and the content of human urinary kallidinogenase in the ulinastatin is particularly and effectively decreased.According to the method, the total yield can be increased to 75% or above, and the product specific activity reaches 4000 U / mg.pr.

Description

technical field [0001] The invention belongs to the technical field of medicine, and in particular relates to a method for purifying ulinastatin based on a hydrophobic column. Background technique [0002] Ulinastatin, also known as Urinary trypsin inhibitor (UTI for short), is a glycoprotein composed of 143 amino acids isolated and purified from human urine, with an isoelectric point of about 2.6 and a molecular weight of about 67,000Da. It has the activity of inhibiting hydrolytic enzymes such as various proteases, carbohydrases and lipases; thereby inhibiting the excessive release of inflammatory mediators; improving microcirculation and tissue perfusion. And when the body is severely injured, it can inhibit the occurrence and development of systemic inflammatory response (SIRS), then block multiple organ dysfunction (MODS), and protect organ functions. [0003] There are more than 300 kinds of proteins in human urine. At present, urokinase, human urinary trypsin inhibit...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/81C07K1/20
CPCC07K14/8114
Inventor 王旭郑少亮许文勤肖益热
Owner GUANGDONG TECHPOOL BIO-PHARMA CO LTD
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