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Pancreatic kallidinogenase purifying process

A pancreatic kininogenase and process technology, applied in the field of separation and purification, can solve the problems of low automation, poor reproducibility, and small scale, and achieve the effects of high automation, shortened time, and good reproducibility

Inactive Publication Date: 2015-12-23
CHANGZHOU QIANHONG BIOPHARMA
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is: the present invention solves the problems of small scale, poor reproducibility and low degree of automation in the prior art by providing a pancreatic kininogenase purification process. Purify pancreatic kininogenase by affinity chromatography to obtain pancreatic kininogenase with high unit titer, high specific activity and high yield

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] A process for purifying kallikrein, comprising the following steps:

[0025] (1) Extraction: add 800kg of crude kallikrein and 10.8 tons of drinking water to the reaction tank, stir until completely dissolved, to obtain a crude extract, adjust the pH to 3.0 with oxalic acid solution, and stir to obtain a reaction after 2 hours of centrifugation clear night;

[0026] (2) Ultrafiltration: pass the clear liquid obtained in step (1) through an ultrafiltration membrane, the specification of the ultrafiltration membrane is a separation molecular weight of 10,000 Daltons, during which purified water is added in stages until the conductivity of the ultrafiltrate reaches 2000 μs / cm collect ultrafiltrate;

[0027] (3) salting out: add ammonium sulfate to ultrafiltrate by mass ratio of ultrafiltrate and ammonium sulfate at 1:0.1, and centrifuge to collect supernatant after standing for 1 hour;

[0028] (4) Chromatography: 200 liters of affinity fillers are loaded into the chroma...

Embodiment 2

[0032] A process for purifying kallikrein, comprising the following steps:

[0033] (1) Extraction: add 800kg of crude kallikrein and 10.8 tons of drinking water to the reaction tank, stir until completely dissolved, to obtain a crude extract, adjust the pH to 4.5 with oxalic acid solution, and stir to obtain the reaction after 2 hours of centrifugation clear night;

[0034] (2) Ultrafiltration: pass the clear liquid obtained in step (1) through an ultrafiltration membrane, the specification of the ultrafiltration membrane is a separation molecular weight of 10,000 Daltons, during which purified water is added in stages until the conductivity of the ultrafiltrate reaches 1600 μs / cm collect ultrafiltrate;

[0035] (3) salting out: add ammonium sulfate to ultrafiltrate by mass ratio of ultrafiltrate and ammonium sulfate 1:0.2, and collect supernatant by centrifugation after standing for 1 hour;

[0036] (4) Chromatography: 300 liters of affinity fillers are loaded into the chr...

Embodiment 3

[0040] A process for purifying kallikrein, comprising the following steps:

[0041] (1) Extraction: add 800kg of crude kallikrein and 10.8 tons of drinking water into the reaction tank, stir until completely dissolved to obtain a crude extract, adjust the pH to 6.0 with an oxalic acid solution, and centrifuge to obtain after stirring for 2 hours clear night;

[0042] (2) Ultrafiltration: pass the clear liquid obtained in step (1) through an ultrafiltration membrane, the specification of the ultrafiltration membrane is a separation molecular weight of 10,000 Daltons, and purified water is added in stages during this period until the conductivity of the ultrafiltrate reaches 1200 μs / cm collect ultrafiltrate;

[0043] (3) salting out: add ammonium sulfate to ultrafiltrate by mass ratio of ultrafiltrate to ammonium sulfate 1:0.5, and centrifuge to collect supernatant after standing for 1 hour;

[0044] (4) Chromatography: 260 liters of affinity fillers are loaded into the chroma...

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PUM

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Abstract

The invention discloses a pancreatic kallidinogenase purifying process. The process includes the steps of extraction, ultrafiltration, salting-out, chromatographic analyzing, ultrafiltration concentration and sterilization freeze-drying. Compared with the prior art, the pancreatic kallidinogenase purifying process has the advantages that the process can meet needs of industrialized large-scale production, is high in reproducibility and can guarantee quality of products different in production batch; by the process, time of the whole production process is reduced, and automation level is high; by the process, 0.35 billion units of pancreatic kallidinogenase products can be obtained in each batch, purity of the products reaches greater than 90% while specific activity of the same is higher than 800U / mg.pro, and time consumed in the whole chromatographic process is shortened to be within 4h.

Description

technical field [0001] The invention relates to a separation and purification method, in particular to a purification process of pancreatic kallikrein. Background technique [0002] Kallikrein is a glycoprotein composed of 18 amino acids and 4 sugars. It acts on the protein substrate kininogen to release the hormone kinin. It has the following effects: dilate blood vessels, improve microcirculation; activate plasmin, reduce blood viscosity; activate phospholipase A2, prevent platelet aggregation, prevent thrombosis, etc.; therefore, it is often used as a clinical vasodilator. In addition, it can also be used to treat microcirculation disorders, such as nephropathy caused by diabetes, peripheral neuropathy, retinopathy, fundus disease and ischemic cerebrovascular disease, and can also be used for the adjuvant treatment of essential hypertension. [0003] Kallikrein is extracted and purified from the pancreas of mammals. The extraction route is to extract from the pancreas i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N9/64
CPCC12N9/6424C12Y304/21035
Inventor 王轲周翔潘伟忠
Owner CHANGZHOU QIANHONG BIOPHARMA