Deoxygenation method and preparation process for natural hemoglobin blood substitute

A blood substitute and hemoglobin technology, applied in the field of biopharmaceuticals, can solve the problems of easy destruction of natural hemoglobin, no deoxygenation method that provides protein structure, and low MetHb content, and achieves simple and routine operation, guaranteed activity and curative effect, and low toxicity. Effects of side effects

Active Publication Date: 2017-09-08
BLOOD TRASFUSION INST CHINESE ACAD OF MEDICAL SCI
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  • Claims
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AI Technical Summary

Problems solved by technology

This method can only ensure that the content of methemoglobin is less than 5%, cannot avoid the toxic and side effects caused by MetHb, and still does not provide a fast and effective deoxygenation method in the purification stage of natural hemoglobin without destroying the protein structure
[0009] The cross-linked and modified hemoglobin has the characteristics similar to red blood cells, and it is not easy to destroy it by conventional deoxygenation methods. However, the structure of natural hemoglobin is easily destroyed by conventional processing methods. Therefore, the preparation of hemoglobin from natural sources is thorough and rapid, and the MetHb content Low blood substitutes present great challenges

Method used

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  • Deoxygenation method and preparation process for natural hemoglobin blood substitute
  • Deoxygenation method and preparation process for natural hemoglobin blood substitute
  • Deoxygenation method and preparation process for natural hemoglobin blood substitute

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Embodiment 1

[0036] Natural hemoglobin purification steps: at 4-20°C, wash human umbilical cord blood with normal saline to obtain 400ml of packed red blood cells, add 1000ml of sterile, pyrogen-free distilled water that has been deoxygenated by high-purity nitrogen through a titanium rod with a pore size of 1μm in advance Membrane rupture for 15 minutes, then add 9g of sodium chloride to make the solution system an isotonic system; add 3% VC to adjust the pH of the solution to 6.0, and add VC to a final concentration of 0.5%, add glucose protectant to a final concentration of 3%, Hb The final concentration is 5%. Continue to vacuumize for 15 minutes and pass high-purity nitrogen through the titanium rod for 15 minutes alternately for more than 3 times to fully deoxidize, and take samples at 1 hour, 2 hours, and 3 hours to measure the PO 2 , SO 2 and MetHb. Then heat the deoxidized Hb at 60°C for 10 hours, cool it to 4°C, and then filter it with a 1 μm water-based microporous membrane, a...

Embodiment 2

[0047] Cross-linking step: at 4°C, adjust the pH of the physiological saline system containing purified Hb obtained in Example 1 to 7.5 with VC, add VC to a final concentration of 0.3%, add sucrose to a final concentration of 3%, and the final Hb concentration to 5.5%. Continue to evacuate for 15 minutes and pass high-purity nitrogen through titanium rods with a pore size of 1 μm for 15 minutes alternately, repeat more than 3 times to fully deoxidize, and take samples at 1 hour, 2 hours, and 3 hours to measure the PO 2 , SO 2 and MetHb, the results are shown in Table 3. Then 1% glutaraldehyde was added to the purified Hb at a rate of 3 ml / min, and after 30 minutes of reaction, 20-fold molar ratio of sodium borohydride was added to terminate the reaction. Finally, after filtering through a 1 μm water-based microporous membrane, the polymerized Hb is obtained by ultrafiltration with an ultrafiltration membrane bag with a molecular weight cut-off of 100KD to remove impurities a...

Embodiment 3

[0051] Modification step: at 4°C, add the polymerized Hb obtained in Example 2 into 0.1M Bis-tris solution, adjust the pH to 8.0 with VC, add VC to a final concentration of 0.3%, and add a glucose protectant to a final concentration of 3% , the final concentration of Hb was 6.4%, and the volume was 2000ml. Continue to vacuumize for 15 minutes and pass high-purity nitrogen through titanium rods with a pore size of 1 μm for 15 minutes to alternately, repeat more than 3 times to fully deoxidize, and take samples at 1 hour, 2 hours, and 3 hours to measure the PO 2 , SO 2 and MetHb, the results are shown in Table 4. Then put the solution in a water bath at 25°C, add phytic acid (15mM), react for 1 hour, add bis(3,5-dibromosalicyl)fumarate (DBBF) in a molar amount 6.0 times of hemoglobin, and react for 2 hours, The reaction was terminated by adding sodium borohydride in an amount 20 times the molar amount of hemoglobin, and the temperature was lowered to 4°C. Finally, after filte...

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Abstract

The invention discloses a deoxygenation method for a natural hemoglobin blood substitute. The deoxygenation method comprises the following steps: adding a small molecule antioxidant and a sugar protective agent into an isotonic system containing natural hemoglobin; adjusting a pH value to 5.0 to 8.0; and alternatively vacuumizing and introducing high-purity inert gas for multiple deoxidization. The invention further discloses a preparation process for the natural hemoglobin blood substitute by adopting a deoxygenation technology. According to the deoxygenation method disclosed by the invention, thorough deoxygenation is realized by alternatively vacuumizing and introducing the gas for the multiple times; before deoxygenation, a proper amount of small molecule antioxygen is added for avoiding the phenomenon that the content of MetHb is increased caused by oxygen introduction before and after deoxygenation; compared with a method of deoxdation by only introducing the gas in the subsequent step in the prior art, the method disclosed by the invention has the advantages that deoxygenation is more thorough and quicker; the content of the hemoglobin is less than or equal to 1 percent, or even is as low as 0 percent; and in addition, a proper amount of sugar protectant is added before deoxygenation for sufficiently protecting a natural hemoglobin structure from being damaged in the deoxygenation process, so that activity and curative effect of a final product are ensured.

Description

technical field [0001] The invention belongs to the field of biopharmaceuticals, and in particular relates to a deoxygenation method and preparation process of a natural hemoglobin blood substitute. Background technique [0002] Blood is the vehicle that delivers nutrients to tissues and removes waste products from them. Blood is composed of red blood cells (RBC or erythrocytes), white blood cells (WBC), platelets, and plasma. Red blood cells make up approximately 99 percent of the cells in the blood, and their main function is to transport oxygen and remove carbon dioxide from tissues. The reversible oxygenation function of red blood cells (i.e. transport of oxygen) is carried out by hemoglobin. Mammalian hemoglobin has a molecular weight of about 64,000 Daltons and is composed of about 6% heme and 94,070 globins. Its native form contains two pairs of subunits (ie, it is a tetramer), each containing a heme group of the scleuzin polypeptide chain. Hemoglobin is in equili...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A61K38/42A61K47/26A61K47/22A61P7/08
CPCA61K38/42A61K47/22A61K47/26
Inventor 李燊周文涛杨成民王红刘嘉馨
Owner BLOOD TRASFUSION INST CHINESE ACAD OF MEDICAL SCI
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