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Process for preparing cross-linked haematoglobin as substitute of red blood cell

A technology of hemoglobin and substitutes, applied in the field of cross-linked hemoglobin, which can solve the problems of high blood pressure, failure to achieve effect, short half-life, etc.

Inactive Publication Date: 2003-02-12
天津协和生物科技发展有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because its half-life is too short and it is easy to cause kidney damage, it cannot be used as a substitute for red blood cells
In addition, because uncross-linked and polymerized hemoglobin molecules can pass through the blood vessel wall and combine with the vasodilator factor NO, resulting in vasoconstriction and increased blood pressure

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0022] The pyridoxal hemoglobin solution modified with PLP by pyridoxal 5-phosphate in a conventional manner is used as raw material, the hemoglobin concentration is 11.26% (w / v), and the methemoglobin content is 3.59% (w / w). In an ice bath at 4°C, adjust the pH to 6.70 with 10% vitamin C solution, at this time the concentration of hemoglobin is 10.6%, and the volume is about 755 ml. Feed wet high-purity N 2 For 0.5-1 hour, the hemoglobin is converted into deoxygenated hemoglobin, 0.1% glutathione is added to the solution as an antioxidant, and lysine is pre-added with 5 times the molar amount of hemoglobin. Under sufficient and gentle agitation, add 1% (w / v) glutaraldehyde solution through the sand core sampling device with a constant flow pump at a rate of 0.1 ml / min·100 ml, and the glutaraldehyde and hemoglobin molecules The molar ratio is 13:1, adding in two times, adding 11 parts for the first time, after reacting for ~0.5 hours, then adding the remaining amount of glut...

Embodiment 2

[0024] The operation mode of the cross-linking reaction is the same as the above example. Wherein, the hemoglobin solution is 650 milliliters of the hemoglobin solution that has been purified but not modified by PLP, the hemoglobin concentration is 12%, and the pH is adjusted to 6.70 with vitamin C. In a 4°C ice bath with humidified N 2 Flow deoxygenation for 0.5-1 hour. Add 2% vitamin C or glutathione before the cross-linking reaction, and glycine or ethanolamine with 5 times the molar weight of hemoglobin as cross-linking competition inhibitors, add 1% glutaraldehyde after deoxygenation treatment, and the addition amount is hemoglobin 15 times the molar weight, the addition rate was 0.2 ml / min·100 ml, and the stirring was continued. After 4 hours, use lysine with 11 times the molar amount of hemoglobin as a terminator, and react for 2 hours. Then 59 ml of 1.56% sodium borohydride solution was added for reduction reaction for 4 hours, conventional filtration and ultrafiltr...

Embodiment 3

[0026] Operation process is with embodiment 1.

[0027] The PLP-modified pyridoxal hemoglobin solution is used as a raw material, the hemoglobin concentration is 8%, the volume is 600 ml, and the pH is adjusted to 6.40 with vitamin C. After pre-adding the arginine solution which is 2 times the molar weight of hemoglobin, add 52.5 milliliters of glutaraldehyde with a concentration of 1%, which is 7 times the molar weight of hemoglobin. The adding speed is 0.3 ml / min·100 ml, and the reaction is 30 minutes. Then, adjust the pH=6.90 with 20% disodium hydrogen phosphate, and after pre-adding 4 times the amount of arginine solution, add 26 milliliters of 1% glutaraldehyde 3.5 times the amount, and the adding speed is the same as before. After the second reaction, adjust the pH=7.40 with the same disodium hydrogen phosphate, and then add 4 times the amount of arginine solution, then add the same glutaraldehyde solution of 3.5 times the amount for the third time, and the speed of add...

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Abstract

A process for preparing the cross-linked haematoglobin used as the substitute of red blood cell includes such steps as deoxygenating haematoglobin solution, regulating pH=6.0-7.5, mixing with polyamino compound as crosslinking competition inhibitor, dispersing glutaraldehyde in it, reaction, adding the said competition inhibitor, reduction reaction, and filtering and ultra-filtering.

Description

technical field [0001] The present invention relates to a process for the preparation of cross-linked hemoglobin for red blood cell substitutes. Specifically, it is a chemical cross-linking treatment method based on hemoglobin, especially human hemoglobin, in order to make it more suitable for use as a substitute for red blood cells. Background technique [0002] Hemoglobin, the natural oxygen-transporting component of the blood, is currently the main target for the production of red blood cell substitutes. It is now known that hemoglobin is a tetrameric structure composed of four subunits, including two α subunits and two β subunits, each subunit has a globulin peptide chain, tetrameric The molecular weight of the bulk is approximately 64,000 Daltons, with each subunit having a similar molecular weight. Studies have found that tetrameric hemoglobin is easily dissociated into αβ dimers after leaving red blood cells, and in some cases, it can even be further dissociated int...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61P7/06C07K14/805
Inventor 杨成民王红杨晓明张鸿辉王雁峰刘嘉馨谢成莲李强余璟
Owner 天津协和生物科技发展有限公司
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