JPH05178900A - Production of powdery hemoglobin - Google Patents

Abstract

PURPOSE:To obtain the subject compound for oxygen transporting agent, artificial blood, etc., capable of suppressing increase of ratio of methemoglobin formation, free from increase of osmotic pressure and viscosity by bringing hemoglobin solution into contact with CO to form coordination complex of hemoglobin and CO and subjecting the coordination complex to lyophilization. CONSTITUTION:A hemoglobin solution taken out from human erythrocyte is put in a vial and the vial is sealed with rubber plug and an injection needle is thrust from a rubber plug of vial bottle therein and the bottle is cooled with ice and carbon monoxide gas excluding oxygen is introduced into the bottle and brought into contact with hemoglobin to form a coordination complex of the hemoglobin and carbon monoxide and a reduction type nicotine amide adeninedinucleotide (NADH) is added to the resultant CO-bound hemoglobin solution and the solution is frozen with liquid nitrogen under nitrogen atmosphere and subjected to lyophilization under reduced pressure to provide the objective powdery hemoglobin suppressed to <=4% in increase of ratio of methemoglobin formation and free from increase of osmotic pressure and viscosity.

Description

Detailed Description of the Invention

[0001]

BACKGROUND OF THE INVENTION 1. Field of the Invention The present invention relates to a powder of oxygen carrier, for example, hemoglobin used as a raw material for artificial blood.

[0002]

2. Description of the Related Art Conventionally, sugars and amino acids have been added to a hemoglobin solution at a concentration of 3% or more, and oxygen bound to hemoglobin has been desorbed by vacuum deaeration or bubbling of nitrogen or argon gas. A method of freeze-drying a body hemoglobin (deoxyHb) solution [eg, Surgery Gynecology Obstetrics, 148, 69-75 (1979);
Biopolymers, 22, 2367-2381 (1983); Cryobiology, 25,
277-284 (1988); Haematologia, 18, 45-52 (1985) and JP-A-61-1620].

However, in these methods, it is necessary to add a large amount of sugars and amino acids in order to prevent denaturation of hemoglobin. When the concentration of additives such as sugars and amino acids is low, suppression of methemoglobin formation can not be expected so much, and on the contrary, even if the concentration of additives is too high, the solution physical properties of the redispersed hemoglobin solution change significantly, for example, osmotic pressure. There is a drawback that the viscosity and the viscosity increase remarkably. Especially when redispersing to prepare a high concentration hemoglobin solution,
This problem becomes more prominent. When preparing hemoglobin used as a raw material for artificial blood, the inclusion of a large amount of sugars and amino acids not only causes an increase in osmotic pressure and viscosity, but also acts as an impurity itself, which is not desirable.

[0004]

The object of the present invention is to overcome the drawback of the addition of a large amount of sugars and amino acids, which has been a problem in the above-mentioned conventional method, and effectively suppress the progress of methemoglobin formation. Powdered hemoglobin can be easily operated,
Moreover, it is to provide a method that can be mass-produced and is advantageous for industrial implementation.

[0005]

Means for Solving the Problems As a result of intensive studies to achieve the above-mentioned object, the present inventors have found that a central metal (heme), which is a pigment of hemoglobin, is brought into contact with carbon monoxide to form a coordination compound. Or, if necessary, a reducing agent for methemoglobin, such as reduced nicotinamide adenine dinucleotide (NADH), ascorbic acid, etc., is added to the hemoglobin solution, and the solution is lyophilized to give hemoglobin. It was found that the physicochemical performance and biological activity of sucrose did not change, and the present invention was completed based on this finding.

That is, the present invention is a method for producing powdered hemoglobin, which comprises the steps of bringing carbon monoxide into contact with a hemoglobin solution to form a coordination complex of hemoglobin and carbon monoxide, and then freezing and drying the same. And optionally a reducing agent is added before the freeze-drying step.

The hemoglobin solution used in the present invention can be prepared by a known method (edited by the Chemical Society of Japan, sequel biochemistry experiment course,
Volume 8, "Blood", Tokyo Kagaku Dojin, 1987; Methods in
Enzymology, Vol. 76, 1981, Academic Press, New Yo
rk; The Chromatography of Hemoglobin, 1983, Dekker
New York; Japanese Patent Laid-Open No. 2-503676, etc.). Erythrocytes are human or cow, horse,
Any animal origin such as pig can be used. Specifically, erythrocytes are washed with a physiological saline using a continuous centrifuge to remove mixed leukocytes and the like. A hypotonic solution such as distilled water is added to the washed red blood cells to perform hemolysis. Erythrocyte membrane substances and blood group substances are removed by filtering the hemolyzed blood with several kinds of membrane filters having pore sizes of 1.0 μm to 0.05 μm. If necessary, concentration is carried out by ultrafiltration to adjust the hemoglobin concentration.

In the step of preparing the hemoglobin solution used in the present invention, an alkaline solution or a buffer solution is added before concentration,
A hemoglobin solution having a pH of 5.5 to 8.0 and an osmotic pressure of 50 to 300 mOsm can be prepared. Coordination between hemoglobin and carbon monoxide is performed by introducing carbon monoxide into a hemoglobin solution and bringing them into contact with each other. The reaction is preferably performed at 0 to 37 ° C, more preferably 0 to 10 ° C. At a temperature of 0 ° C. or lower, the fluidity of the hemoglobin solution is reduced and the solution becomes gelled, which is not suitable because the formation of the coordinator is significantly delayed. Further, if the temperature exceeds 37 ° C, denaturation such as oxidation of hemoglobin is promoted, which is not desirable. The purity of carbon monoxide is not particularly limited, but a concentration of 50% or more is desirable. If it is less than 50%, the reaction efficiency is deteriorated, which is not desirable. It may contain an inert gas, for example, impurities such as nitrogen and argon. Oxygen contained in carbon monoxide does not have to be removed, but in order to increase the reaction efficiency, a deoxidizer (for example, 1 g of sodium dithionite and
It is preferred to use carbon monoxide gas deoxygenated by passing 10 mg of anthraquinone-β-sulfonic acid in 100 ml of distilled water). The progress of the reaction can be confirmed by the absorption at 419 nm and 569 nm in the UV spectrum that appears when a coordination between hemoglobin and carbon monoxide is formed.

As the reducing agent for methemoglobin (hereinafter simply referred to as reducing agent), reduced nicotinamide adenine dinucleotide (NADH), cytochrome b ₅ reductase system, ascorbic acid, glutathione, etc. can be used. It is desirable that the reducing agent is dissolved in distilled water or a buffer solution in advance and then added to hemoglobin in a solution state. The reducing agent solution is preferably stored at a temperature of -40 to 10 ° C. The concentration of the reducing agent added to the hemoglobin solution is 0.1 to 50 mM, preferably 1.0 to 20 mM.

The reducing agent may be added either before or after the formation of a coordination body of hemoglobin and carbon monoxide, but in order to suppress the production of methaemoglobin during the formation of the coordination body, the reducing agent is added. Is preferably added before the formation of the coordination body with carbon monoxide.

In the present invention, the hemoglobin solution in which the coordination with carbon monoxide is formed can be frozen at -10 ° C or lower, for example, liquid nitrogen, a dry ice / methanol freezing agent, or a low temperature freezer. You may use the method of. The freezing operation is preferably carried out under an atmosphere of an inert gas such as nitrogen or argon. Using a rotary evaporator or other device, swirl the flask containing the hemoglobin so that the solution is evenly dispersed on the inner wall of the device and frozen. Then, the flask was attached to a freeze dryer, and the internal temperature was -30 to -85 ° C,
Freeze-drying is performed at a vacuum degree of 1 to 100 mtorr, preferably 1 to 10 mtorr. Care should be taken to maintain the outside temperature at 0 to 25 ° C, preferably 10 to 20 ° C during freeze-drying. The water content of the powdered hemoglobin obtained by controlling the drying time can be adjusted to 15% or less. It takes 5 to 24 hours depending on the treatment amount and the water content of the desired product. The red powdered hemoglobin obtained is preferably stored in the dark at 4 ° C.

For redispersion of the powdered hemoglobin in the present invention, as a solvent, for example, distilled water or a buffer solution having a pH of 5.5 to 8.0 such as phosphate, hydrogen carbonate, Tris salt and HEPES is used. Distilled water or buffer can be added to powdered hemoglobin to redisperse hemoglobin by incubation at 0-15 ° C. Redispersion can be promoted by applying stirring or vibration as necessary.
The obtained hemoglobin solution can be sterilized by a method such as filter filtration, if necessary.

[0013]

EFFECTS OF THE INVENTION According to the method of the present invention, the amount of methaemoglobin (metHb) produced is extremely low in the operation of powdering hemoglobin, and the increase in the meteoric ratio (metHb%) can be suppressed to 4% or less. In addition, the obtained powdered hemoglobin does not contain extra sugars and amino acids, and thus the drawbacks of the conventional method such as changes in the physical properties of the redispersion solution, such as an increase in osmotic pressure and viscosity, can be overcome. The obtained powdered hemoglobin is extremely suitable as a raw material for artificial blood.

[0014]

EXAMPLES The present invention will be described in more detail with reference to Examples and Comparative Examples. For the measurement of various properties such as the concentration of the hemoglobin solution and the rate of methemoglobin, a Model 288 fully automatic pH / blood gas electrolyte analyzer, Model 2500 CO oximeter (manufactured by Ciba Corning) was used. A hemox analyzer (manufactured by TCS Medical Products) was used to measure the oxygen bond-dissociation curve. The osmotic pressure was measured using an ONE-TEN type Osmometer (manufactured by Fiske Corp.). The viscosity was measured using a rotational viscometer Bio-Rheolyzer (manufactured by Tokyo Keiki Co., Ltd.), the viscosity measurement temperature was 37 ° C., and the rotation speed of the viscometer was 10 rpm.

Example 1 A hemoglobin solution having a concentration of 16.0 g / dl extracted from a human erythrocyte that had expired (osmotic pressure 289 mOsm, viscosity 1.55 mPa xs) 100 m
l was placed in a 250 ml vial and sealed. An injection needle was inserted from the rubber stopper of the vial bottle, cooled with ice, and oxygen-free carbon monoxide gas was introduced to convert hemoglobin into a carbon monoxide coordinator (CO hemoglobin).
The progress of the reaction with carbon monoxide was confirmed by the absorption of UV spectra at 419 nm and 569 nm, and the reaction was completed in about 15 minutes.

20 ml each of the obtained CO-hemoglobin solution was transferred to four 250 ml eggplant type flasks, and NADH was added to each flask so as to have a concentration of 0, 2.0, 5.0 and 10.0 mM. Under a nitrogen atmosphere, the flask was immersed in liquid nitrogen while rotating, and the CO-hemoglobin solution was uniformly frozen so as to adhere to the wall of the device in a film form. Attach the flask to a lyophilizer (Vertis, 25SL type), -85 ℃,
Lyophilized at 2-10 mtorr. The outside temperature was kept at 17 to 20 ° C. After freeze-drying for 20 hours, red powdered hemoglobin was obtained.

Distilled water was added to each of the obtained powdery hemoglobins having different amounts of added NADH to adjust the total volume to 20 ml. Hemoglobin was redispersed by gentle shaking at 4 ° C. The hemoglobin concentration of the solution, the methoterization rate (metHb%), the percentage of the carbon monoxide coordination compound in the total hemoglobin concentration (COHb%), pH, osmotic pressure, and viscosity were measured. Table 1 summarizes the performance such as the methation rate of the hemoglobin solution in which the powdered hemoglobin is redispersed and the hemoglobin solution before pulverization. Also, before pulverization (dotted line)
And the corresponding hemoglobin solution (NADH) after pulverization (solid line)
The UV spectrum at a concentration of 2 mM) is shown in FIG. From the analysis of the spectrum, denaturation of hemoglobin after powdering (increase in methemoglobin) was hardly observed.

[0018]

[Table 1]

Example 2 Concentrations 31.6, 15.8 and 7.9 g prepared in the same manner as in Example 1
20 ml of each / dl hemoglobin solution was placed in each of three vials, and NADH was added to each vial at 2.0 mM. In the same manner as in Example 1, the solution was brought into contact with carbon monoxide to CO-convert hemoglobin, and then frozen with liquid nitrogen,
Lyophilization was performed at -85 ° C and 2 to 10 mtorr for 24 hours. A red powdered hemoglobin was obtained. For redispersion of the powdered hemoglobin, a phosphate buffer solution (concentration: 10 mM) having a pH of 7.32 was used, and the hemoglobin was redispersed in the same manner as in Example 1.
The concentration of the obtained hemoglobin solution, the methation ratio, etc. were measured and are shown in Table 2.

[0020]

[Table 2]

Example 3 80 ml of a hemoglobin solution (Tris salt buffer, pH 7.33) having a concentration of 10.3 g / dl taken out from the expired human red blood cells was used to form a carbon monoxide coordination compound by the method of Example 1. After the NADH
Was added to 3 mM. The solution was transferred to a 1000 ml flask and freeze-dried for 24 hours in the same manner as in Example 1.
g of red powdered hemoglobin was obtained. Distilled water was added to 10 g of the obtained powdery hemoglobin (containing Tris salt, NaCl, etc.) to adjust the volume of the solution to 20 ml. Four
After gently stirring at ℃ for 15 minutes, a hemoglobin solution having a concentration of 37.5 g / dl, a methotization rate of 4.5%, a pH of 7.36 and an osmotic pressure of 378 mOsm was obtained.

Carbon monoxide bound to hemoglobin was desorbed by blowing oxygen gas for 1 hour while irradiating the solution with white light of 60 W under ice cooling. The formation of oxygenated coordinator of hemoglobin (oxyhemoglobin) was confirmed by visible spectrum absorption at 415 nm. According to a conventional method, the oxygen transport performance of the solution was measured with a hemox analyzer. The oxygen bond-dissociation curves of hemoglobin before pulverization (dotted line) and after pulverization (solid line) are shown in FIG. As shown in FIG. 2, it was clarified that the oxygen carrying performance hardly changed.

Example 4 Ascorbic acid was added at a concentration of 25 mM to 80 ml of a hemoglobin solution (osmolarity: 187 mOsm) having a concentration of 16.0 g / dl taken out from the expired human red blood cells, and then monoxide was added in the same manner as in Example 1. A carbon coordination complex was formed. The solution was transferred to a 1000 ml flask and freeze-dried for 42 hours as in Example 1. Redispersion was performed in the same manner as in Example 1. The resulting hemoglobin solution had a concentration, a rate of methemoglobin and an osmotic pressure of 14.5 g / dl, 5.5%, and 205 mOsm, respectively.

Comparative Example 1 Carbon monoxide coordination compound (CO-containing Hb) of hemoglobin of Example 1
Was lyophilized in the same manner as in Example 1 except that the deoxyhemoglobin (deoxyHb) was used instead of the above. 20 ml of 16.0 g / dl hemoglobin solution was put into a 250 ml flask, and the bound oxygen was desorbed by blowing nitrogen gas.
Deoxy hemoglobin was used. The production of deoxy-hemoglobin was confirmed by the absorption around 430 nm in the UV spectrum. After the liquid was frozen in liquid nitrogen under a nitrogen atmosphere, freeze-drying and other operations were carried out according to Example 1. 2.78
g of brown powdered hemoglobin was obtained. When the obtained powder was redispersed in distilled water, insoluble matter was observed. The properties of the redispersed hemoglobin solution are shown in Table 3. Metho-hemoglobin (metHb) was 55.4%.

Comparative Example 2 NAO was added to the deoxy-hemoglobin solution obtained in Comparative Example 1.
Lyophilization was performed according to the procedure of Example 1 except that DH was added at a concentration of 2 mM. When the obtained powder was redispersed in distilled water, insoluble matter was observed. The properties of the redispersed hemoglobin solution are shown in Table 3. Methemoglobin (metHb)
It was 49.0%.

Comparative Example 3 NAO was added to the deoxy-hemoglobin solution obtained in Comparative Example 1.
Lyophilization was performed according to the procedure of Example 1 except that sucrose was added at a concentration of 4% instead of DH. A red powdered hemoglobin was obtained. Distilled water was added to the whole obtained powdery hemoglobin to adjust the total volume to 20 ml. Hemoglobin was redispersed by gentle shaking at 4 ° C. The properties of the redispersed hemoglobin solution are shown in Table 3. Methaemoglobin (me
tHb) was 7.9%, the osmotic pressure of the solution was 405 mOsm, and the viscosity was 2.19 mPa xs. The hemoglobin solution obtained had high osmotic pressure and viscosity, and was unsuitable as a raw material for artificial blood.

[0027]

[Table 3]

[Brief description of drawings]

FIG. 1 is a UV spectrum of hemoglobin before and after powderization. In the figure, the solid line is the spectrum after pulverization and the dotted line is the spectrum before pulverization.

FIG. 2 is an oxygen bond-dissociation curve of hemoglobin before and after pulverization. In the figure, the solid line is the graph after pulverization and the dotted line is the graph before pulverization.

Continued Front Page (72) Inventor Hidetoshi Tsuchida 2-10-10 Sekimachi Minami, Nerima-ku, Tokyo

Claims (2)

[Claims] 1. A method for producing a powdered hemoglobin, which comprises contacting carbon monoxide with a hemoglobin solution to form a coordinator of hemoglobin and carbon monoxide, and then freeze-drying this. 2. The method for producing powdered hemoglobin according to claim 1, wherein a reducing agent for methaemoglobin is added before the freeze-drying step.