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Improved polymerized hemoglobin preparation method

A technology of hemoglobin and deoxyhemoglobin, which is applied in the direction of hemoglobin/myoglobin, chemical instruments and methods, specific peptides, etc., can solve the problems of high cost, cumbersome steps, and long preparation cycle, and achieve long solution cycle and high effective conversion rate Effect

Inactive Publication Date: 2014-01-22
BLOOD TRASFUSION INST CHINESE ACAD OF MEDICAL SCI
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Although these methods can achieve certain good results, they all have the disadvantages of long preparation period, cumbersome steps, and high cost.

Method used

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  • Improved polymerized hemoglobin preparation method
  • Improved polymerized hemoglobin preparation method
  • Improved polymerized hemoglobin preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0027] Measure 100ml of stromal-free human placental hemoglobin liquid with a free concentration of 5.5% at 4°C (prepared in normal saline, the same below), and add it to 150ml of N2-filled 2 Put the suction filter bottle in the ice bath, pass nitrogen for about 30 minutes, so that the hemoglobin is in a deoxygenated state; put the suction filter bottle with hemoglobin on the magnetic stirrer, the stirring speed is 750rpm, continue to pass nitrogen for 20 minutes -25min, add glutaraldehyde by adding sample through titanium rod, so that the molar ratio of glutaraldehyde to hemoglobin is 5.5 : 1, 30min sampling, and add a certain amount of NaBH 4 The solution, after no air bubbles are generated, is sealed and placed in a refrigerator at 4°C for detection. The result is as figure 1 as shown in a.

[0028] figure 1 It is the HPLC schematic diagram of the influence of different concentration hemoglobin solution raw materials on the preparation method of the present inventio...

Embodiment 2

[0032] Measure 100ml of stromal-free human placental hemoglobin liquid with a free concentration of 6.0% at 4°C, and add it to 150ml of N2-filled 2 Place the suction filter bottle in an ice bath, pass nitrogen for about 30 minutes, so that the hemoglobin is in a deoxygenated state; place the suction filter bottle containing hemoglobin above a magnetic stirrer, stir at 600 rpm, and continue to pass nitrogen for 20-25 minutes, Add glutaraldehyde by feeding through a titanium rod so that the molar ratio of glutaraldehyde to hemoglobin is 6.5 : 1. Take a sample at 90 minutes and add a certain amount of NaBH 4 The solution, after no air bubbles are generated, is sealed and placed in a refrigerator at 4°C for detection. see results figure 2 as shown in c.

[0033] figure 2 It is an HPLC schematic diagram of the influence of different cross-linking agents and hemoglobin molar ratios on the preparation method of the present invention. Wherein, 2a is the HPLC figure of lower mol...

Embodiment 3

[0035] Take 100ml of stroma-free human placental hemoglobin solution with a free concentration of 6.0% at 4°C, and add it to 150ml of N2-filled 2 Place the hemoglobin in a suction filter bottle at 6°C for about 30 minutes to keep the hemoglobin in a deoxygenated state; put the suction filter bottle with hemoglobin on top of a magnetic stirrer at a stirring speed of 500 rpm, and continue to pass nitrogen for 20- 25min, add glutaraldehyde by adding sample through titanium rod, so that the molar ratio of glutaraldehyde to hemoglobin is 6.0 : 1. Take a sample at 60 minutes and add a certain amount of NaBH 4 The solution, after no air bubbles are generated, is sealed and placed in a refrigerator at 4°C for detection. The result is the same as figure 2 Consistent with that shown in b.

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Abstract

The invention relates to an improved polymerized hemoglobin preparation method. According to the invention, a hemoglobin aqueous solution with matrix-free human deoxyhemoglobin weight / volume content of 4-14% is adopted as a raw material. Under an inert gas environment and a stirring condition, an aldehyde-type bi-functional crosslinking agent is delivered into the raw material solution through a titanium rod; a polymerization reaction is carried out under a temperature of 0-10 DEG C, such that polymerized hemoglobin with an average molecular weight of 100-210kD and a preferable molecular weight of 110-170kD is obtained. A molar ratio of hemoglobin to the aldehyde-type bi-functional crosslinking agent is 4-8:1. With the method, the polymerized hemoglobin product with relatively low average molecular weight, relatively narrow molecular weight distribution, and effective conversion rate reaching 69% can be prepared, such that a problem of non-uniform molecular weight distribution in product of current polymerized hemoglobin preparation is effectively avoided. The operation is simple, and equipment cost is low.

Description

technical field [0001] The invention relates to a method for preparing polymerized hemoglobin, in particular to a method for preparing polymerized hemoglobin with lower average molecular weight, narrower molecular weight distribution and higher effective conversion rate. Background technique [0002] Since stroma-free hemoglobin cannot be directly applied to the human body, hemoglobin oxygen carriers (HBOCs) have become the mainstream research direction in the field of international blood substitutes. In recent decades, people mainly improve the safety of HBOCs products and prolong their action time in vivo by polymerizing hemoglobin and modifying pyridoxal pentaphosphate (PLP). After decades of research, breakthroughs have been made in this field of technology, and some research products in the United States, Canada and other countries have completed phase III clinical trials. However, with the application of HBOCs in experimental animals or clinical studies, problems of v...

Claims

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Application Information

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IPC IPC(8): C07K14/805
CPCC07K14/805
Inventor 周文涛杨成民王劲峰刘嘉馨李燊王红李凤娟王翔陈刚
Owner BLOOD TRASFUSION INST CHINESE ACAD OF MEDICAL SCI
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