Looking for breakthrough ideas for innovation challenges? Try Patsnap Eureka!

A kind of preparation method of high-purity trypsin

A trypsin, high-purity technology, applied in the field of preparation of high-purity trypsin, can solve the problems of weak trypsin purification ability, low product titer, long time consumption, etc., to improve market competitiveness, low chymotrypsin content, increase quality effect

Active Publication Date: 2017-11-24
SPH NO 1 BIOCHEM & PHARMA CO LTD
View PDF2 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The problem to be solved by the present invention is to overcome the technical defects of the existing process of trypsin, such as weak purification ability, long time consumption, and low titer of the finished product, and provide a preparation method of high-purity trypsin

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0035] (1) Crude product dissolution: Add 1.2kg of trypsin crude product to 6000mL pH2.8 aqueous solution and stir to dissolve;

[0036] (2) Inactivation of virus by low pH incubation method: add 5mol / L HCl 14mL, adjust the pH of the crude trypsin solution to pH 3.1, and place it at 20-25°C for 1 hour;

[0037] (3) Hydrophobic chromatography purification: Pack the hydrophobic chromatography filler phenyl-Sepharose (purchased from GE Healthcare) into a column of 20×16 cm, and use pH7.050mmol / L Na 2 HPO 4 -NaH 2 PO 4 , 1mol / L (NH 4 ) 2 SO 4 The buffer solution is used as a balance solution to balance, and the solution in step (2) is taken to load the sample. The processing method of the sample solution: 5mol / L NaOH to adjust the pH to pH7.0, 4°C, 4000rpm / min, centrifuge for 20min, and the sample is loaded Rinse the column with the above equilibrium solution until peak A flows through 280nm Down to the baseline, then with pH7.050mmol / L Na 2 HPO 4 -NaH 2 PO 4 Solution e...

Embodiment 2

[0044] (1) Crude product dissolution: Add 1.2kg of trypsin crude product to 6000mL pH2.8 aqueous solution and stir to dissolve;

[0045] (2) Inactivation of virus by low pH incubation method: add 5mol / L HCl 17mL, adjust the pH of the crude trypsin solution to pH 2.9, and place it at 20-25°C for 1 hour;

[0046] (3) Hydrophobic chromatography purification: Hydrophobic chromatography filler phenyl-Sepharose (purchased from GE Healthcare) was packed into a column of 20 × 16 cm, and pH 8.050 mmol / L Na 2 HPO 4 -NaH 2 PO 4 , 1mol / L (NH 4 ) 2 SO 4 The buffer solution is used as a balance solution to balance, and the dissolved solution in step (2) is taken to load the sample. The treatment method of the sample solution is 5mol / L NaOH to adjust the pH to pH8.0, centrifuge at 8°C and 4000rpm / min for 15min, and the sample is loaded. Rinse the column with the above equilibrium solution until peak A flows through 280nm Down to the baseline, then with pH8.050mmol / L Na 2 HPO 4 -NaH ...

Embodiment 3

[0053] (1) Crude product dissolution: Add 1.2kg of trypsin crude product to 6000mL pH2.8 aqueous solution and stir to dissolve;

[0054] (2) Inactivation of virus by low pH incubation method: add 5mol / L HCl 17mL, adjust the pH of the crude trypsin solution to pH 2.9, and place it at 20-25°C for 1 hour;

[0055] (3) Hydrophobic chromatography purification: Hydrophobic chromatography filler phenyl-Sepharose (purchased from GE Healthcare) was packed into a column of 20 × 16 cm, and pH7.5 50mmol / L Na 2 HPO 4 -NaH 2 PO 4 , 1mol / L (NH4) 2 SO 4 The buffer solution is used as a balance solution to balance, and the solution in step (2) is taken to load the sample. The treatment method of the sample solution is 5mol / L NaOH to adjust the pH to pH7.5, centrifuge at 4°C and 4000rpm / min for 15min, and the sample is loaded. Rinse the column with the above equilibrium solution until peak A flows through 280nm down to baseline, then with pH7.5 50mmol / L Na 2 HPO 4 -NaH 2 PO 4 Solution...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

A preparing method of high-purity trypsin is disclosed. The preparing method includes following steps: (1) dissolving a crude product, (2) inactivating viruses by a low-pH incubation method, (3) purifying by hydrophobic chromatography, (4) performing zymogen activation, (5) purifying by affinity chromatography, (6) desalting an eluate, (7) performing membrane filtration to remove the viruses and (8) performing vacuum freeze-drying to obtain the high-purity trypsin. According to the preparing method, the hydrophobic chromatography is adopted to purify the zymogen of the trypsin, the trypsin is purified by the affinity chromatography after the zymogen activation, a process is simple and convenient, and the high-purity high-titer trypsin can be obtained.

Description

technical field [0001] The invention relates to the technical field of biochemical drug pharmacy, in particular to a preparation method of high-purity trypsin. Background technique [0002] Trypsin is an important proteolytic enzyme secreted by animal pancreas, which can hydrolyze proteins into polypeptides or amino acids. It has selective hydrolysis of peptide chains of arginine and lysine, and is an endopeptidase. Trypsin can digest pus and necrotic tissue, so that normal tissue can secrete serum to remove foreign matter and promote the growth of new granulation. It has a wide range of applications, including: 1. Local edema, hematoma, abscess, etc. caused by various ulcers, inflammatory gangrene, traumatic injury, etc.; 2. Bronchitis, bronchial asthma, dry cough, etc. ); 3. All kinds of snake bites, etc. [0003] In the prior art, the preparation of trypsin has been using multiple salting-out, crystallization and dialysis processes for a long time. This process has wea...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N9/76
CPCC12N9/6427C12Y304/21004
Inventor 黄臻辉汪雅雯周伟洁
Owner SPH NO 1 BIOCHEM & PHARMA CO LTD
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Patsnap Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Patsnap Eureka Blog
Learn More
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic, Popular Technical Reports.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap|About US| Contact US: help@patsnap.com