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Blood substitute prepared by using hematoglobin-human seralbumin conjugate and its preparation method

A technology of human serum albumin and hemoglobin, applied in the field of blood substitute research, can solve problems such as limitations, short retention time, and large gap in blood function

Inactive Publication Date: 2004-10-13
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

Some side effects of blood substitutes (such as vasoconstriction and high blood pressure) limit their use, especially in the elderly with various medical conditions
Secondly, the product has a short retention time in the internal circulation (as short as a few hours, the longest life span is 70 hours, while the life span of natural red blood cells is 120 days), it can only provide short-term oxygen supply function, and it is different from normal blood function. big gap

Method used

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  • Blood substitute prepared by using hematoglobin-human seralbumin conjugate and its preparation method
  • Blood substitute prepared by using hematoglobin-human seralbumin conjugate and its preparation method
  • Blood substitute prepared by using hematoglobin-human seralbumin conjugate and its preparation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0033] Embodiment 1: Preparation of pure matrix-free bovine hemoglobin by electrophoresis

[0034] Take a certain volume of washed fresh bovine blood red blood cells, and use twice the volume of cold swelling solution (20mmol / L KH containing 0.6% NaCl 2 PO 4 / Na 2 HPO 4 (PBS) buffer, pH 7.4) suspension, shake at 4°C for 1 h; pump 20 mmol / L PBS buffer (pH 7.4) twice the volume of red blood cells at a rate of 10% of the total volume of the solution per minute, shake for 1 h and then adjust The concentration of NaCl salt is 0.9%, and the red blood cell swelling and rupture solution is obtained. The rupture fluid was pretreated by a Millipore Pellicon cross-flow membrane filtration system. First, use 0.22μm membrane microfiltration to remove cell debris and macromolecular impurities, and then use membrane ultrafiltration with a molecular weight cut-off of 10KD to remove small molecular impurities. The obtained hemoglobin solution was further purified by DEAE Sepharose Fast Fl...

Embodiment 2

[0035] Embodiment 2: Preparation of chromatographically pure matrix-free porcine hemoglobin

[0036] Take a certain volume of washed fresh porcine red blood cells, and use twice the volume of cold swelling solution (20mmol / L KH containing 0.6% NaCl 2 PO 4 / Na 2 HPO 4 (PBS) buffer, pH 7.4) suspension, shake at 4°C for 1 h; pump 20 mmol / L PBS buffer (pH 7.4) twice the volume of red blood cells at a rate of 10% of the total volume of the solution per minute, shake for 1 h and then adjust The concentration of NaCl salt is 0.9%, and the red blood cell swelling and rupture solution is obtained. The rupture fluid was pretreated by a Millipore Pellicon cross-flow membrane filtration system. First, use 0.45μm membrane microfiltration to remove cell debris and macromolecular impurities, and then use membrane ultrafiltration with a molecular weight cutoff of 30KD to remove small molecular impurities. The resulting hemoglobin solution was further purified by Q Sepharose Big Beads ani...

Embodiment 3

[0037] Example 3: One-step cross-linking of human hemoglobin and human serum albumin by m-maleimide benzoic acid-N-hydroxysuccinimide ester (MBS)

[0038] Since the sulfhydryl group of the HSA molecule will react with MBS, thereby affecting the coupling reaction. Therefore, blocking of remaining sulfhydryl groups is required prior to activation of HSA with MBS. Slowly add 0.2 ml of 30 mM iodoacetamide to 10 ml of 5 mg / ml HSA solution (HEPES buffer at pH 7.8), and react at room temperature for 20 minutes. Subsequently, 0.5 ml of 30 mM MBS solution (dissolved in dimethylformamide) was added and reacted at room temperature for 30 minutes. The reaction mixture was passed through a Sephadex G-25 gel filtration column to remove excess MBS and iodoacetamide, and the protein fraction was collected. Because the hemoglobin molecule has a reactive thiol group (the sulfhydryl group of β-93 cysteine), the activated HSA can directly react with the hemoglobin molecule. The collected prote...

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Abstract

The present invention provides a hemoglobin and human serum albumin conjugate and its preparation method. It adopts membrane filtration and ion exchange chromatographic method to purity hemoglobin, and adopts liquid phase one-step coupling method, two-step coupling methoe or method capable of making protein be adsorbed on the solid phase medium to make hemoglobin and human serum albumin implement coupling reaction to obtain conjugate. Said conjugate is undergone the processes of ion exchange chromatography, ultrafiltration and gel filtration chromatographic purification to make the molecular weight distribution of said conjugate be in the between of 100 KD-300 KD. Said product has good characteristics of blood substitute.

Description

technical field [0001] The invention relates to a blood substitute, in particular to the research field of blood substitutes with hemoglobin as the substrate. Background technique [0002] Blood transfusion is an indispensable medical method for clinical operations, disaster relief and battlefield rescue. Relying on people to donate blood not only faces the problem of shortage of blood sources, but also due to the complexity of blood types, blood transfusion must be strictly matched. Security is threatened. In recent years, the demand for blood has been increasing, but safe and effective blood sources have become increasingly scarce. Therefore, in recent decades, human blood substitutes have always been a research and development focus in the international scientific and business circles. [0003] Human blood substitutes, that is, human red cell substitutes, are artificial preparations that have the function of oxygen transfer, maintain blood osmotic pressure and acid-bas...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/42C07K14/765C07K14/805
CPCC07K14/805A61K38/42C07K14/765A61P7/08
Inventor 苏志国路秀玲郑春杨徐宇红胡涛
Owner INST OF PROCESS ENG CHINESE ACAD OF SCI
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