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Purification method of four blood-derived proteins

A protein and source technology, applied in the field of purification of four blood-derived proteins, can solve problems affecting subsequent use, unfavorable amplification, and low activity, and achieve the effects of strong stability, low cost, and high product purity

Active Publication Date: 2021-02-26
ZYBIO INC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] With the advancement of modern medicine, the demand for ATT, TRF, HP, and AAG is increasing day by day. However, due to their different properties, if they are purified individually in serum, there will be problems such as low purity, low yield, and unfavorable amplification.
It is difficult to purify multiple proteins at the same time with the existing technology, because the activity of proteins treated with organic solvents or high salt is often low, which seriously affects subsequent use

Method used

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  • Purification method of four blood-derived proteins
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  • Purification method of four blood-derived proteins

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0092] Example 1: Separation and purification of HP, AAG, AAT, and TRF from human blood.

[0093] The filler used in anion exchange chromatography is: UniCore-DEAE, and the filler used in cation exchange chromatography is: UniGel-80SP.

[0094] 1. Dialyze the sample human serum into a chromatographic buffer (pH=4.7) to obtain a dialysis sample, and use anion exchange chromatography to purify the dialysis sample; the chromatographic buffer is: 10-50mM sodium acetate buffer, pH= 4.7, equilibrate the anion exchange chromatography column with chromatography buffer, then use gradient elution with 0-100% elution reagent A, the pH of 0-100% elution reagent A is 4.7, collect flow-through solution I and wash Eluent I: Under the condition of pH=4.7, the flow-through I contains two proteins of TRF and AAT, and the eluate I contains two proteins of HP and AAG.

[0095] 2. Equilibrate the HP affinity chromatography column with 20mM PBS, load the eluent I to the HP affinity chromatography ...

Embodiment 2

[0104] Example 2: Separation and purification of AAT, AAG, HP, and TRF from human blood.

[0105] The filler used in anion exchange chromatography is: UniGel-30Q, and the filler used in cation exchange chromatography is: UniGel-30SP.

[0106] 1. The sample human serum is dialyzed into a chromatographic buffer (pH=5.0) to obtain a dialysis sample, and the dialysis sample is purified by anion exchange chromatography; the chromatographic buffer is: 10-50mM sodium acetate buffer, pH=5.0 5.0, equilibrate the anion exchange chromatography column with chromatographic buffer, then use 0-100% elution reagent A gradient elution, the pH of the 0-100% elution reagent A is 5.0, collect the flow-through solution I and eluate I; under the condition of pH=5, the flow-through I contains TRF protein, and the eluate I contains AAT, HP and AAG three proteins.

[0107] 2. Adjust the pH of the flow-through solution I to 5.3, and purify it with cation exchange chromatography packing. The chromatogr...

Embodiment 3

[0119] The filler used in anion exchange chromatography is: NanoGel-50Q, and the filler used in cation exchange chromatography is: UniCore-CM.

[0120] 1. Dialyze the sample human serum into a chromatographic buffer (pH=4.5) to obtain a dialysis sample, and use anion exchange chromatography to purify the dialysis sample; the chromatographic buffer is: 10-50mM sodium acetate buffer, pH= 4.5, equilibrate the anion exchange chromatography column with chromatography buffer, then use 0-100% elution reagent A gradient elution, the pH of the 0-100% elution reagent A is 4.5, collect the flow-through solution I and eluate I; under the condition of pH=4.5, the flow-through I contains two proteins of HP and AAG, and the eluate I contains two proteins of AAT and TRF.

[0121] 2. Equilibrate the affinity chromatography column with 20mM PBS, load the flow-through solution I to the HPT affinity chromatography column for purification, collect the flow-through solution II that flows out first,...

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Abstract

The invention discloses a preparation method of four blood-derived proteins. The method adopts a purification method combining anion / cation exchange chromatography and affinity chromatography, and four proteins, namely alpha1-antitrypsin (AAT), transferrin (TRF), alpha1-acidic glycoprotein (AAG) and haptoglobin (HP) are purified from a same blood sample. The preparation method is simple to operate, the obtained target protein is stable in activity, high in purity, high in yield and suitable for industrial production and clinical application.

Description

technical field [0001] The invention relates to the separation and purification of proteins, in particular to the purification method of four blood-derived proteins. Background technique [0002] In the era of rapid development of biomedicine, the blood product industry has become an important part of the biomedicine and diagnostic industry. At present, plasma-derived blood products (plasma protein drugs) mainly include albumin, immunoglobulin, coagulation factors, and protease inhibitors. Blood product companies in developed countries can obtain more than 20 kinds of plasma protein drugs from plasma. However, limited by factors such as talent, technology, and clinical cognition, domestic blood product companies can only obtain no more than 10 products, which are mainly separated and purified based on low-temperature ethanol precipitation, including human serum albumin, immunoglobulin Protein and coagulation factor VIII products, etc. At present, the main purification of p...

Claims

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Application Information

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IPC IPC(8): C07K14/81C07K14/79C07K14/47C07K1/22C07K1/18C07K1/14
CPCC07K14/8125C07K14/79C07K14/473C07K14/47
Inventor 易维京朱攀任燕斌赵威赵忠颢
Owner ZYBIO INC
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