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A kind of preparation method of polymerized hemoglobin with low polymer content

A technology of hemoglobin and inert gas is applied in the field of preparation of polymerized hemoglobin with low polymer content, which can solve the problems of affecting hemodynamic characteristics and tissue oxygen supply capacity, reducing the effective utilization rate of hemoglobin, and large molecular weight of polymerized hemoglobin, etc. The risk of pollution and endotoxin exceeding the standard, the risk of reducing renal toxicity and vasoconstriction, the effect of easier application

Active Publication Date: 2022-04-19
REDPHARM BEIJING BIOPHARMACEUTICAL INST CO LTD +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Because although glutaraldehyde is the most commonly used homobifunctional cross-linking agent for the preparation of polymerized hemoglobin, glutaraldehyde also has many disadvantages: it has two aldehyde groups, so it has high reactivity; it has a long carbon chain, It is conducive to the occurrence of intermolecular cross-linking, making the molecular weight of polymerized hemoglobin too large; there is no specific reaction site, and there are about 40 lysine side chain amino groups and four chain terminal amino groups on the surface of hemoglobin that can participate in the reaction, making polymerization difficult to control
This wide molecular weight distribution not only reduces the effective utilization rate of hemoglobin, but also has poor stability if the molecular weight is too low, it is easy to dissociate and cause nephrotoxicity, and the oxygen carrying efficiency is low.
Although high average molecular weight does not change the oxygen-carrying properties of polymerized hemoglobin, it reduces the oncotic pressure and increases the viscosity of plasma, which in turn affects its hemodynamic properties and tissue oxygen supply capacity

Method used

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  • A kind of preparation method of polymerized hemoglobin with low polymer content
  • A kind of preparation method of polymerized hemoglobin with low polymer content
  • A kind of preparation method of polymerized hemoglobin with low polymer content

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preparation example Construction

[0237] 2.3 Preparation of buffer required for detection:

[0238] Prepare buffer 750mM MgCl as follows 2 , 50 mM tris, 0.1 mM EDTA pH 6.5.

[0239] 2.3.1 Measure 800mL of ultrapure water with a 1000mL graduated cylinder and transfer it to a 1L beaker.

[0240] 2.3.2 Add 10.46±0.05g of Bis-Tris to a 1L beaker and stir gently with a magnetic stirring bar until completely dissolved.

[0241] 2.3.3 Adjust the pH of the solution to 6.7±0.1 with concentrated hydrochloric acid, and stir gently.

[0242] 2.3.4 Add 152.48±0.05g of MgCl to the 1L beaker 2 6H2O.

[0243] 2.3.5 Add 0.030±0.001g of EDTA to a 1L beaker and stir gently with a magnetic stirring bar until it is completely dissolved.

[0244] 2.3.6 Adjust the pH of the solution to 6.5±0.1 with concentrated hydrochloric acid, and stir gently.

[0245] 2.3.7 Adjust the pH to the required range, use a 1000ml graduated cylinder, and increase the volume to 1000ml with ultrapure water.

[0246] 2.3.8 Filter the solution using ...

Embodiment 1

[0266] The preparation of embodiment 1 cross-linked hemoglobin

[0267] like figure 1 As shown, dilute the deoxygenated hemoglobin to 2g / dL, add NAC with a final concentration of 3mg / mL, and move it into the anaerobic reaction vessel 12. The height of its liquid level 10 should not exceed half of the height of the anaerobic reaction vessel 12. The pressure atomizer 1 is fixed on the top of the oxygen reaction vessel 12, the inlet pipe 3 and the liquid inlet pipe 2 are connected to the atomizer 1, and the balance gas pipeline 4 is connected to the top of the anaerobic reaction vessel 12 to adjust the internal pressure of the anaerobic reaction vessel 12. required pressure, close the anaerobic reaction vessel 12, open the bottom stirring rotor 11, heat the reaction solution to 42°C through the water bath jacket 13, open the liquid inlet pipe valve 5, add the cross-linking agent glutaraldehyde, adjust the liquid inlet speed to 260mL / min, open the inlet pipe valve 8, feed an in...

Embodiment 2

[0270] The preparation of embodiment 2 cross-linked hemoglobin

[0271] like figure 1 As shown, dilute the deoxygenated hemoglobin to 3g / dL, add NAC with a final concentration of 3mg / mL, and move it into the anaerobic reaction vessel 12. The height of its liquid level 10 should not exceed half of the height of the anaerobic reaction vessel 12. The pressure atomizer 1 is fixed on the top of the oxygen reaction vessel 12, the inlet pipe 3 and the liquid inlet pipe 2 are connected to the atomizer 1, and the balance gas pipeline 4 is connected to the top of the anaerobic reaction vessel 12 to adjust the internal pressure of the anaerobic reaction vessel 12. required pressure, close the anaerobic reaction vessel 12, open the bottom stirring rotor 11, heat the reaction solution to 42°C through the water bath jacket 13, open the liquid inlet pipe valve 5, add the crosslinking agent glutaraldehyde, adjust the liquid inlet speed to 500mL / min, open the inlet pipe valve 8, feed an ine...

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Abstract

The invention relates to a method for preparing polymerized hemoglobin with low polymer content, and specifically provides a method for preparing cross-linked hemoglobin, including: a. diluting deoxygenated hemoglobin; b. cross-linking agent glutaraldehyde and the diluted deoxygenated hemoglobin Linking reaction; c, add reducing agent to terminate crosslinking reaction, remove unreacted crosslinking agent and stop reducing agent with the mode of ultrafiltration changing liquid, obtain crosslinked hemoglobin; Wherein: in described step a, dilute deoxygenated hemoglobin concentration to 1-3g / dL, add N-acetyl-L-cysteine ​​in described hemoglobin, make the mass ratio of hemoglobin and N-acetyl-L-cysteine ​​in the solution be 5-10:1; And hemoglobin The mass ratio to the crosslinking agent is 1000:29‑45. In the polymerized hemoglobin prepared by the method, not only the dimer content is low, but also the high polymer content is also low.

Description

[0001] priority information [0002] This patent application claims the priority of the Chinese patent application 201910846580.9 with the application date of September 9, 2019 and the title of the invention is "Preparation method of cross-linked hemoglobin and organ perfusion fluid containing the hemoglobin". The priority application is hereby cited in its entirety Reference. technical field [0003] The invention belongs to the field of biopharmaceuticals, and relates to a polymerization method of hemoglobin, in particular to a preparation method of polymerized hemoglobin with low polymer content. Background technique [0004] Blood transfusion is an essential medical method for surgical operations, emergency rescue and disaster relief, and battlefield rescue. In recent years, the world's demand for blood for transfusion has grown exponentially. However, due to the complex blood types of human blood, transfusion reactions are always difficult to overcome, and the storage...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C07K14/805C07K14/415C07K19/00A01N1/02
CPCC07K14/805C07K14/415C07K5/0215A01N1/0247C07K2319/00
Inventor 游可为史国营张彦鹏石松陈浩源
Owner REDPHARM BEIJING BIOPHARMACEUTICAL INST CO LTD
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