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Process for preparing uniform molecule weight polymerized hemoglobin

A technology of hemoglobin and molecular weight, which is applied in the field of biomedicine, can solve the problems of expensive cross-linking agent, difficult to buy, uneven molecular weight distribution of polymers, etc., and achieve the effect of overcoming the unevenness of polymerization products and the reduction of colloid osmotic pressure

Inactive Publication Date: 2003-05-28
INST OF PROCESS ENG CHINESE ACAD OF SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

But these crosslinkers are expensive and not readily available, and the molecular weight distribution of the polymer is still not uniform

Method used

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  • Process for preparing uniform molecule weight polymerized hemoglobin
  • Process for preparing uniform molecule weight polymerized hemoglobin
  • Process for preparing uniform molecule weight polymerized hemoglobin

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] Example 1 Preparation of polymerized bovine hemoglobin with uniform molecular weight using Q Sepharose fast flow medium:

[0024] Fill the pure bovine hemoglobin solution with nitrogen at 4°C for 2 hours to make the bovine hemoglobin in a deoxygenated state; equilibrate the Q Sepharose fast flow medium with 20mM HEPES buffer (pH 8.0); pump to remove the oxygen in the Q Sepharose fast flow medium , then filled with nitrogen; take 15ml of treated solid phase and 10ml of deoxygenated hemoglobin (3mg / ml, pH8.0) and evenly mix; Glutaraldehyde solution was reacted at 4°C for 1 hour under nitrogen protection; then equilibrated with 10 column volumes of 20mM HEPES buffer (pH8.0); finally, with 20mM HEPES buffer (pH8.0) containing 0.4M sodium chloride .0) elution, the eluate was detected at 280nm, and the elution peak was collected carefully; after the collection was reduced with sodium borohydride, the salt was removed through a dextran G-25 column; the collection was placed on...

Embodiment 2

[0025] Example 2 Preparation of polymerized bovine hemoglobin with uniform molecular weight using Q agarose large particle medium:

[0026] The hemoglobin solution was filled with nitrogen at 4°C for 2 hours to make the hemoglobin in a deoxygenated state. The Q Sepharose macroparticle medium was equilibrated with 20 mM HEPES buffer (pH 8.0). Aspirate to remove oxygen from the Q Sepharose medium and flush with nitrogen. Take 15ml of the treated solid phase and mix evenly with 5ml of 6mg / ml deoxyhemoglobin (pH8.0). Subsequently, it was loaded into a 6.5×1 cm column, 0.5 ml of 0.4% (v / v) glutaraldehyde solution was added, and the mixture was reacted at 4° C. for 1 hour. Subsequently, equilibrate with 10 column volumes of 20 mM HEPES buffer (pH 8.0) at a flow rate of 1 ml / min. Finally, it was eluted with 20 mM HEPES buffer (pH 8.0) containing 0.4 M sodium chloride at a flow rate of 0.6 ml / min, and the eluted peaks were collected. After the collection was reduced with sodium bo...

Embodiment 3

[0027] Example 3 Preparation of polymerized hemoglobin with DEAE agarose fast flow medium:

[0028] Take 10 ml of 3 mg / ml hemoglobin solution (pH 8.0) and fill it with nitrogen gas at 4°C for 2 hours to make the hemoglobin in a deoxygenated state. The DEAE Sepharose fast flow medium was equilibrated with 20 mM HEPES buffer (pH 8.0). Aspirate to remove oxygen from the DEAE agarose fast flow medium. Take 15ml of processed DEAE agarose fast flow medium and mix with deoxygenated hemoglobin. The mixture was loaded into a 6.5×1 cm column, 0.5 ml of 0.4% (v / v) glutaraldehyde solution was added, and reacted at 4° C. for 1 hour under nitrogen protection. It was then equilibrated with 10 column volumes of 20 mM HEPES buffer (pH 8.0). Finally, it was eluted with 20 mM HEPES buffer (pH 8.0) containing 0.4 M sodium chloride, paying attention to collecting the elution peak. After the collection was reduced with sodium borohydride, it was passed through a Sephadex G-25 column to remove t...

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Abstract

A process for preparing the poly-haematoglobin with homogeneous molecular weight includes adsorbing haematoglobin by solid phase, adding dual-function reagent, polymerizing reaction, eluting and collecting the polymer by buffering liquid, and chromatography for separating and purifying the polyhaematoglobin. Its advantages are homogeneous molecular weight (about 135 thosands Dol), easy separation, and lower osmotic pressure.

Description

field of invention [0001] The invention belongs to the field of biomedicine, in particular to a method for preparing polymerized hemoglobin with uniform molecular weight. Background technique [0002] Hemoglobin consists of four subunits (α 1 alpha 2 beta 1 beta 2 ), a protein in red blood cells responsible for oxygen transport. Because of its unique oxygen binding characteristics, normal physiological metabolic pathways and other reasons, hemoglobin-based blood substitutes are very in line with the physiological needs of the human body. Unlike red blood cells, blood substitutes can be sterilized by pasteurization, ultrafiltration and chemical methods, thereby removing pathogenic microorganisms that cause AIDS and hepatitis B. Because there is no red blood cell group antigen, there is no need for cross blood testing, saving time and reducing the requirements for equipment. This allows blood transfusions to be administered as quickly as a sal...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C07K14/805C08H1/00
Inventor 苏志国胡涛
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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